Download 22nd EMC Full Program - 25th Enzyme Mechanisms Conference

22nd Enzyme Mechanisms Conference
ABSTRACTS
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O1
Application of Kinetic and Structural Insights for Enzyme-Inhibitor
Interactions in Aurora Kinase Drug Discovery
John R. Pollard
Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon,
Oxfordshire, UK, OX14 4RY
The Aurora kinases are a family of three homologous proteins that each play critical roles in
mitosis and cell division. Amplification of Aurora genes and overexpression of these proteins
has been observed in many cancers and in certain cases this has been associated with
chromosomal instability, a common hallmark of cancer. Furthermore, overexpression of
Aurora proteins can confer a cancer-like phenotype on some normal cells. Consequently these
kinases have been viewed as attractive targets for anti-cancer drug discovery efforts.
A wealth of structural and kinetic data for both Aurora-A and -B has provided great insight
into the mechanism of action for these kinases. Most notably this knowledge has enabled a
number of very elegant structure-based design approaches to deliver a series of clinical
candidates with diverse biochemical properties1.
VX-680 is competitive for ATP with high affinity for all three Aurora kinases and was the
first of the Aurora inhibitors to enter the clinic. Although highly selective for the Aurora
kinases the compound potently and unexpectedly cross-reacted with wild type and refractory
mutant forms of Abl and Flt kinases, all of which have limited sequence homology in the
ATP pocket with the Aurora proteins.
A series of extended kinetic and structural studies have shown that VX-680 binds to Aurora-B
with time-dependent kinetics that are characterised by a slow off-rate. These data are
consistent with a two-step binding mode and the formation of a tight-binding complex of VX680 with an inactive form of the enzyme. Notably, a hydrophobic pocket that appears critical
for the tight binding of VX-680 in the inactive Aurora-B conformation is conserved in both
the Abl and Flt enzyme structures, which could explain the observed cross-reactivity profile.
A comprehensive understanding for the mechanism of inhibition of VX-680 provides a strong
base for the rational design of second generation inhibitors that both exploit beneficial crossreactivity profiles and augment the time-dependent nature of inhibition, capitalising on a
potential pharmacodynamic effect.
The talk will include a detailed description of the kinetic and structural characteristics of
Aurora inhibition by VX-680, how the mechanism of inhibition influences cellular properties
and how a detailed knowledge of ligand-enzyme interactions can influence rational drug
discovery efforts.
References
1. Discovery and Development of Aurora Kinase Inhibitors as Anticancer Agents Pollard J.
R., Mortimore M., J. Med. Chem 2009, 52, 2629-2651
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O2
Enzymatic Analysis of Normal and Oncogenic Receptor Tyrosine Kinase
Signaling Impacting Drug Design
Jim Solowiej1, Sergei Timofeevski1, Phillip Schwartz1, Simon Bergqvist1, Jingrong Cui2,
Helen Zou1, Tami Marrone2, Kevin Ryan2, Robert Kania2, James Christensen1, Gordon
Alton1, Wade Diehl2, Michele McTigue2, Brion W. Murray1
1
Pfizer Oncology Research, 10777 Science Center Drive, San Diego, CA 92121
2
Pfizer Chemistry, 10777 Science Center Drive, San Diego, CA 92121
Receptor tyrosine kinases (RTKs) are important cancer drug discovery targets yet the
molecular underpinnings of RTK regulation, function, oncogenic activation, and drug
selectivity are still not fully understood. Oncogenic receptor tyrosine kinases and clinical
agents were studied to further our understanding of this important enzyme class.
Receptor tyrosine kinases such as cMet can be activated by altering the catalytic domain
conformation through an autophosphorylation reaction. cMet is a key regulator in a subset of
cancers, in part, through activating oncogenic mutations which affect downstream signal
transduction. cMet and eight clinically relevant mutants were characterized by biochemical,
biophysical, and cellular methods. A model of RTK activation is proposed to describe how a
RTK response may be matched to a biological context through enzymatic properties. In
addition, two highly optimized cMet inhibitors currently in clinical trials were used to
evaluate the balance between overall kinase selectivity and mutant penetrance.
The catalytic domains of protein kinases are commonly treated as independent modular units
with distinct biological functions. Interactions between regulatory and kinase domains of
VEGFR2 were studied for their effect on kinase biology and inhibitor interactions. The
VEGFR2 ATP-competitive inhibitor axitinib was found to have 40-fold enhanced potency
toward VEGFR2 containing a catalytic domain with a 19 amino acid residue juxtamembrane
region (Ki = 28 pM) relative to the VEGFR2 catalytic domain alone. The larger construct
better correlated better with cellular and in vivo findings. Calorimetric studies confirmed the
potency and provided insight into the thermodynamic origin of the potency differences. A
model was developed from biophysical and structural studies which illuminates a potential
new role for the juxtamembrane domain: an N-terminal/C-terminal catalytic domain clasp
which alters the catalytic domain conformational dynamics.
We’ve begun to study the molecular underpinnings of acquired resistance to EGFR oncology
drugs. Kinetic methods were developed to evaluate tight-binding irreversible inhibition of the
different forms of EGFR (wild-type, activating mutant, acquired resistance double mutant).
Insight from the kinetic analysis is being used to inform effective drug design.
Taken together, these enzymatic studies enable a better understanding of receptor tyrosine
kinase biology and are critical to the design of new therapeutic agents by accounting for
complex biochemical behavior.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O3
Impact of Enzymology and Biophysics on Small Molecule Hit Validation
and Lead Identification
George H. Addona Ph.D.
Merck Research Laboratories, In Vitro Pharmacology Department, West Point, PA 19486
High-throughput screening approaches have resulted in the ability to execute screens on a
wide-variety of target classes. For example, these targets include soluble enzymes, membrane
proteins (enzymes and GPCRs) and protein-protein interactions. Each of these target classes
pose challenges for downstream hit-validation and lead identification and different
approaches are required to identify legitimate small molecule series that inhibit or activate the
selected target. This presentation will focus on approaches taken that increase the probability
of success in identifying and ultimately validating legitimate chemical matter. For instance,
many challenging targets are not amenable to classic detection technologies due to high signal
background, poor sensitivity and/or specificity (undesirable high substrate concentrations
required) and thus require higher-resolution detection modalities enabling proper enzymatic
conditions for meaningful hit discovery. Such technologies include LCMS and other labelfree methods. However, even under optimal conditions that minimize false negatives and
enrich for a pool of hits, every screen is prone to nonspecific inhibition. Therefore, the hit-tolead phase requires a variety of approaches that deconvolute the initial hits. The goal is to
validate hits that are specific, reversible and bind with a stoichiometry consistent with the
mode of action. For soluble targets, direct binding methods offer an excellent ability to
measure kinetics, stoichiometry, effect on protein conformational state as well as mode of
action on hundreds if not thousands of compounds post-screen. Examples will be presented
describing the approach from HTS to validated compounds for these protein families. For
soluble targets that are amenable to structural biology, this approach also provides a key
framework for enabling structure-based design. For membrane enzymes, the challenge is
greater as many biophysical approaches are not amenable to this form of hit-validation. Here
the reliance on optimal screening assays and detailed enzymatic follow-up with the necessary
throughput to support medicinal chemistry will be discussed. Typically, biophysically and/or
enzymatically validated hits result in the ability to advance chemical series quickly and have
demonstrated biological target engagement. Overall, just as assay design is critical for
successful hit-finding equally critical is the hit-to-lead molecular characterization which
forms the foundation for advancement of series to lead optimization.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O4
The rapid catalase activity in the dual function heme enzyme catalaseperoxidase (KatG) requires a radical on its novel Met-Tyr-Trp adduct
Richard S. Magliozzo, Xiangbo Zhao, Javier Suarez, Abdelahad Khajo
Brooklyn College CUNY, Dept. of Chemistry, 2900 Bedford Avenue, Brooklyn, NY 11210 and
The Graduate Center, 365 Fifth Avenue, New York, NY 10016
Catalase-peroxidase (KatG) is a dual-function heme enzyme found in all microorganisms. In
the intracellular pathogen M. tuberculosis, KatG is important for virulence and for activation
of the anti-TB agent isoniazid. No physiological peroxidase substrate has been identified for a
KatG, while explaining its robust catalase activity (2H2O2 = O2 + 2H2O) remains an
interesting challenge especially since its active site is designed more like that of a typical
peroxidase than a monofunctional catalase. All KatGs contain a unique post-translational
modification in the form of a covalent adduct in which the side chains of Met255-Tyr229Trp107 are covalently linked (Fig. 1). Mutagenesis of any residue of the adduct (MYW)
eliminates catalase activity but has no effect on peroxidase function. A transient tyrosyl-like
radical is formed in KatG during turnover of millimolar H2O2 and the heme group is found in
the oxyferrous state during the reaction (at neutral pH). This is unusual because: 1) the
catalase reaction mechanism has not been considered to require a protein-based radical in
heme catalases; and 2) oxyferrous heme is generally a dead-end intermediate in typical
peroxidase reaction pathways. When oxyferrous heme is produced in KatG lacking the
radical, it has the expected high stability. Isotopic labelling of KatG provided evidence for
radical localization on the MYW adduct (Fig. 2) with most spin density residing within the
Tyr-229 ring. An alternate catalase reaction pathway (Fig. 3) in which superoxide anion
dissociates from iron and is quenched by an MYW radical is proposed.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O5
Human frataxin is an allosteric switch that activates the Fe-S cluster
biosynthetic complex
Chi-Lin Tsai, Jennifer Bridwell-Rabb, Andrew Winn, and David Barondeau
Department of Chemistry, Texas A&M University, College Station, TX 77842
Cellular depletion of the human protein frataxin is correlated with the
neurodegenerative disease Friedreich’s ataxia (FRDA) and results in the inactivation of Fe-S
cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe-S
clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that
human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core
machinery for Fe-S cluster biosynthesis. Frataxin binding dramatically changes the KM for
cysteine from 0.59 to 0.011 mM and the catalytic efficiency (kcat/KM) of the cysteine
desulfurase from 25 to 7900 M−1s−1. Oxidizing conditions diminish the levels of both complex
formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine
desulfurase activity. Kinetic analysis of Isu2 variants coupled to mass spectrometry
experiments supports a model in which frataxin functions with Fe2+ as an allosteric activator
that triggers sulfur delivery to Isu2 for Fe-S cluster assembly. Structure-function studies of
FRDA clinical variants reveal a strong correlation between the severity of the clinical
phenotype and loss of the ability to activate the Fe-S cluster assembly complex. We propose a
model1 in which cellular frataxin levels regulate human Fe-S cluster biosynthesis that has
implications for mitochondrial dysfunction, oxidative stress response, and both
neurodegenerative and cardiovascular disease.
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References
1. C. Tsai and D.P. Barondeau “Human Frataxin Is an Allosteric Switch That Activates the
Fe−S Cluster Biosynthetic Complex” Biochemistry (2010) 49:9132–9139.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O6
Computational Approaches to Leverage Enzyme Structures to
Predict Function
Chakrapani Kalyanaraman and Matthew P. Jacobson
Department of Pharmaceutical Chemistry, UCSF, 600 16th St., San Francisco, CA 941582517
Reliable assignment of function to enzymes discovered in genome sequencing projects is a
major challenge in genomic biology. I will discuss the use of computational ligand docking
and protein homology modeling, by our group and others, to help guide investigations of
enzyme-substrate specificity, and the identification of novel enzymatic functions. Our group
has used the functionally diverse enolase superfamily as a model system for testing
approaches to exploiting structure to predict enzyme substrates, in a series of retrospective
and prospective studies. At this point, we have studied hundreds of enzymes in this
superfamily using computational approaches, providing insight into the sequence and
structural determinants of specificity. Enzymatic assays (group of John Gerlt, UIUC) and
crystallography (group of Steve Almo, Albert Einstein College of Medicine) have confirmed
a number of the prospective predictions. Finally, I will discuss the strengths, weaknesses,
and future prospects for this type of “structure-based” approach to investigating protein
function, and the complementarity with “sequence-based” methods.
References
1. C. Kalyanaraman, K. Bernacki, and M. P. Jacobson. "Virtual screening against highly
charged active sites: Identifying substrates of alpha-beta barrel enzymes", Biochemistry,
44 (2005) 2059–2071.
2. L. Song, C. Kalyanaraman, A. A. Fedorov, E. V. Fedorov, M. E. Glasner, S. Brown, P. C.
Babbitt, S. C. Almo, M. P. Jacobson, and J. A. Gerlt. “Assignment and Prediction of
Function in the Enolase Superfamily: A Divergent N-Succinyl Amino Acid Racemase
from Bacillus cereus”, Nature Chemical Biology, 3 (2007) 486–491.
3. C. Kalyanaraman, H. J. Imker, A. A. Federov, E. V. Federov, M. E. Glasner, P. C.
Babbitt, S. C. Almo, J. A. Gerlt, and M. P. Jacobson. “Discovery of a new dipeptide
epimerase enzymatic function guided by homology modeling and virtual screening”,
Structure, 16 (2008) 1668–1677.
4. J. F. Rakus, C. Kalyanaraman, A. A. Fedorov, Elena V. Fedorov, F. P. Mills-Groninger,
K. Bain, J. M. Sauder, S. K. Burley, S. C. Almo, M. P. Jacobson, and J. A. Gerlt.
“Computation Facilitated Assignment of Function in the Enolase Superfamily: A
Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis”,
Biochemistry, 48 (2009) 11546-58.
5. C. Kalyanaraman and M. P. Jacobson. “Studying enzyme-substrate specificity in silico:
A case study of the E. coli glycolysis pathway” (Rapid Report), Biochemistry, 49 (2010)
4003-4005.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O7
Kinetic Cooperativity in a Monomeric Enzyme
Mioara Larion and Brian G. Miller
Department of Chemistry and Biochemistry, 217 Dittmer Laboratory of Chemistry, Florida
State University, Tallahassee, FL 32306-4390
Human glucokinase catalyzes the rate-limiting step of glucose metabolism in the pancreas and
liver. As such, glucokinase is a key regulator of glucose homeostasis. Genetic lesions in glk
result in maturity-onset diabetes of the young (MODY), whereas mutations that enhance
catalysis cause hyperinsulinemia of infancy (HI). Under steady-state conditions glucokinase
activity displays a sigmoidal response to increasing glucose concentrations that is
characterized by a Hill coefficient of 1.7. The existence of kinetic cooperativity in
glucokinase is interesting because the enzyme functions exclusively as a monomer under
physiological conditions. Current models suggest that cooperativity relies upon a slow,
glucose-dependent conformational change that occurs with a rate constant slower the kcat.
Consistent with that postulate, crystal structures of glucokinase confirm that large-scale
structural rearrangements accompany glucose binding. Despite the postulated involvement of
conformational changes in glucokinase cooperativity, no experimental work has been directed
toward understanding the functional dynamics of this enzyme. In this seminar, I will present
an initial investigation of glucokinase dynamics using high resolution NMR, the results of
which offer new insight into the mechanism of kinetic cooperativity in this monomeric
catalyst.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O8
Privileged scaffolds, "underlying" promiscuity, & convergence in the
evolution of new enzyme functions
Patricia Babbitt
University of California, San Francisco, CA 94158
Investigation of structure-function relationships in large and functionally diverse enzyme
superfamilies suggests that many evolve multiple reactions using "privileged" scaffolds,
structural templates whose active site architectures facilitate catalysis of common partial
reactions or other chemical capabilities. Natural evolution has used such scaffolds to evolve
many different reactions and reaction specificities consistent with these functional
capabilities. Using protein similarity networks, global views of these relationships can be
generated quickly for many superfamilies [1], providing new insights about how new
functions evolve and illustrating how an "underlying promiscuity" observed in these
superfamilies can inform the functional assignment of proteins of unknown function and
investigation of their mechanisms [2-4]. This global view also allows us to suggest
convergent evolution of specific enzyme functions from different intermediate ancestors in
the lineage of some of these superfamilies.
References
1. 1.Atkinson HJ, Morris JH, Ferrin TE and Babbitt PC. Using sequence similarity networks
for visualization of relationships across diverse protein superfamilies, PLoS ONE. 4:
e4345, 2009.
2. Atkinson HJ and Babbitt PC. An atlas of the thioredoxin fold class reveals the complexity
of function-enabling adaptations, PLoS Comput Biol. 5: e1000541, 2009.
3. Sakai A, Fedorov AA, Fedorov EV, Schnoes AM, Glasner ME, Brown S, Rutter ME, Bain
K, Chang S, Gheyi T, Sauder JM, Burley SK, Babbitt PC, Almo SC and Gerlt JA.
Evolution of enzymatic activities in the enolase superfamily: stereochemically distinct
mechanisms in two families of cis,cis-muconate lactonizing enzymes, Biochemistry. 48:
1445-1453, 2009.
4. Song L, Kalyanaraman C, Fedorov AA, Fedorov EV, Glasner ME, Brown S, Imker HJ,
Babbitt PC, Almo SC, Jacobson MP and Gerlt JA. Prediction and assignment of function
for a divergent N-succinyl amino acid racemase, Nat Chem Biol. 3: 486-491, 2007.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O9
Insight into enzyme evolution through protein engineering approaches
Simone Eisenbeis, Bettina Schreier, Birte Höcker
Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany
The complex and highly functional proteins that we observe today evolved from simpler and
less specialized subunits. In this process domains as well as subdomains are recruited and
adapted. Since evolution is driven by function, nature has to build on the existing
functionalities of the recruited units. This is a concept that can be adapted to protein
engineering, while at the same time engineering approaches can give insight into how
evolution might have occurred. Here we present two different approaches.
Inspired by the concept of combinatorial assembly we used stable protein fragments to
construct new proteins. We were able to build new βα-barrel proteins from fragments of two
different folds, the flavodoxin-like and the (βα)8-barrel fold. The crystal structure of one of
these proteins revealed the molecular interactions of the fragments and showed that the
fragments retain their structures within the new protein context. It further uncovered the
presence of an additional strand in the inner barrel, which is formed by residues of the Cterminus. This new and unexpected structural element relieves stress at the interface. We then
optimized the interface using computational design with the Rosetta program. Furthermore,
ligand binding was introduced into the protein chimera with only two mutations making use
of an already present phosphate-binding site provided by one of the fragments. Altogether this
modular assembly approach enables new combinations of functional properties encoded in
fold fragments, a mechanism that thus could have easily taken place in the course of evolution
of this typical enzyme fold.
Approaching the question of enzyme evolution from a different perspective, we investigated
the highly specialized catalytic mechanism of enolase. This central enzyme requires two
magnesium ions to facilitate its reaction, while other members of the enolase superfamily, that
convert equally acidic substrates, need only one magnesium ion per active site. To investigate
the importance of the second magnesium ion (MgII), we removed all MgII coordinating
residues and replaced them by positively charged side-chains. High-resolution crystal
structures and activity assays show that the introduced side-chains prohibit MgII binding but
fail to promote catalysis. However, control mutants without positively charged side-chains
retained basal enolase activity through binding of MgII to the substrate in an open active site
without the help of coordinating residues. Thus, we believe that ancestral enolase activity
might have evolved in a member of the enolase superfamily that provided only the necessary
catalytic residues and the binding site for MgI.
References
1. Bharat, Eisenbeis, Zeth & Höcker (2008) A βα-barrel built by the combination of
fragments from different folds. PNAS, 105: 9942-7.
2. Schreier & Höcker (2010) Engineering the enolase magnesium II binding site: implications
for its evolution. Biochemistry, 49: 7582-9.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O10
Dynamic and thermodynamic properties of enzymatic transition states and
complexes with transition state analogues
Steven C. Almo1, Karunesh Arora2, Charles L. Brooks III2, Achelle A. Edwards1, Gary B.
Evans3, Mahmoud Ghanem1, Rong Guan1, Jennifer S. Hirschi1, Meng-Chiao Ho1, Lei Li1,
Minkui Luo1, Andrew S. Murkin1, Suwipa Saen-Oon1, Vern L. Schramm1, Steven D.
Schwartz1, Rafael G. da Silva1, Peter C. Tyler3, Matthew J. Vetticatt1
1
Albert Einstein College of Medicine, Bronx, NY 10461. 2The University of Michigan, Ann
Arbor, MI, 48109. 3Carbohydrate Chemistry Team, Industrial Research Ltd., Lower Hutt,
New Zealand.
Kinetic isotope effects in combination with constrained quantum chemical calculations
provide estimates for structures of enzymatic transition states. Molecular electrostatic
potential maps of the wave function generated for transition states are used to design
transition state analogue inhibitors. Transition state analogues with Km/Kd values above
1,000,000 are common with the current record being 400,000,000. Several transition state
analogues are in various phases of preclinical or clinical development.
Structural, mutational, thermodynamic and computational dynamic analyses of transition state
analogues provide some insights into the nature of enzymatic transition states and the
interaction of transition state analogues with their targets. A few of these properties will be
exemplified with human purine nucleoside phosphorylase (PNP), human methylthioadenosine
phosphorylase (MTAP) and bacterial methylthioadenosine nucleosidase (MTAN).
• Structures of PNP comparing substrate and transition state analogues show multiple new
H-bonds and ionic contacts, readily explaining the increased binding energy.
• Transition state analogue binding to PNP is enthalpy-driven with entropic penalties.
Dynamically active complexes alter entropic penalties and can exhibit tight binding.
• Loss-rates for transition state analogues in vitro and in vivo differ because of recapture.
• Transition state analogue binding to MTAP is entropy-driven.
• PNP and MTAN isozymes with high homology exhibit distinct transition states.
• Transition state structures are sensitive to mutational changes remote from the catalytic
site and dynamic motion is linked to transition state structure.
• Transition state lifetime for PNP is estimated to be 10 fsec with a 70 fsec reaction
coordinate; too fast for thermodynamic equilibrium.
• PNP finds the transition state by a stochastic search process.
• Dynamic motions on the time scale of catalysis (msec) are linked to product release.
• The short PNP transition state lifetime prevents ribocation capture by solvent even when
solvent leaks are introduced into the catalytic site by loop and flap mutations.
• Experimental approaches are in development to perturb fast dynamic motions in PNP.
Transition state analogues are useful in drug development and in understanding catalysis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O11
Structural and Mechanistic Bases for the G:G Specificity of ASFV DNA
Polymerase X
Ming-Daw Tsai
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Our lab has been investigating the kinetic and structural mechanisms of the high-fidelity
mammalian DNA polymerase β (Pol β), and the error-prone viral DNA polymerase X (Pol
X). The goal is to understand how DNA polymerases achieve the high fidelity and the low
fidelity respectively. In this lecture I will report our recent advances for both enzymes. For
Pol β, we have developed and performed global kinetic analyses, which led to uncovering of
the following new mechanistic insight: (i) The well characterized, fast subdomain closing
actually occurs in two steps, both are faster than the chemical step. (ii) The nucleotidyl
transfer (i.e., chemistry) is adequately described by a single reaction step, thus providing
evidence against the existence of additional rate-determining steps prior to chemistry. (iii) pHvariation and [Mg2+]-dependence studies were performed to gain insight into processes
involved in active site reorganization that occurs prior to catalysis of nucleotidyl transfer. For
Pol X, we report the solution structure of Pol X-MgdGTP-DNA ternary complex, which
represents the first solution structure of a DNA polymerase ternary complex, and the first
structure containing a real G:G mismatch. Unlike other polymerases, DNA interacts with
helix E of the fingers subdomain. Structural and kinetic analyses support a Hoogsteen G:dG
(anti-syn) basepairing. Our data further demonstrate that, in contrast to other DNA
polymerases, Pol X pre-binds MgdGTP specifically and productively before binding DNA.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O12
Studies of the Domain Interface in Non-ribosomal Peptide Synthetases
Jesse A. Sundlov1, Daniel J. Wilson2, Ce Shi2,
Courtney A. Aldrich2, and Andrew M. Gulick1
1
Hauptman-Woodward Institute and Department of Structural Biology,
University at Buffalo, Buffalo, NY 14203
and
2
Center for Drug Design, University of Minnesota, Minneapolis, MN 55455
Nonribosomal peptide synthetases (NRPSs) produce a diverse array of peptides that, together
with the functionally-similar polyketide synthases (PKSs), produce many natural products
with important pharmaceutical activity. Many NRPS peptide products alternately function as
siderophores, iron-chelators that are produced in iron-limiting conditions. Because of the low
iron concentrations in relevant biological environments, siderophore biosynthetic pathways
have recently been investigated as targets for inhibition. NRPS and PKS enzymes have been
described as molecular assembly lines, with multiple fused modules each responsible for the
incorporation of one starter unit into the final bioactive product. During synthesis, the nascent
peptide or polyketide is covalently bound to integrated carrier protein domains that deliver the
intermediate to adjacent catalytic domains. This fascinating biosynthesis raises structural
questions regarding the choreography and coordinated delivery of the carrier domains to the
neighboring catalytic domains.
NRPS amino acid substrates are initially activated by an adenylation domain that selectively
loads the pantetheine cofactor of an adjacent carrier domain. To better understand the
structural interface and the conformational rearrangements that guide the NRPS adenylation
and carrier domains, we have engineered a fusion protein between a carrier protein and an
adenylation domain from the enterobactin cluster of E. coli. With the aid of a mechanismbased inhibitor, we have determined the structure of this construct, which forms a dimer that
shows the active interface of two intermolecular transfer steps. The structure gives insight into
the interaction of NRPS domains and the requirements for interdomain linker sequences.
Comparison with other NRPS crystal structures also supports our previous hypothesis that
domain rearrangements within the adenylation domain are mechanistically important to the
NRPS assembly line.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O13
New Activities from an “Old” Enzyme: The 4-Oxalocrotonate
Tautomerase Saga
Christian P. Whitman
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin,
Austin, TX, 78712
4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial
catabolism of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the
conversion of 2-hydroxymuconate (1) to 2-oxo-3-hexenedioate (2), where Pro-1 functions as
a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5
position of product. Two arginine residues, Arg-11 and Arg-39, facilitate the reaction with
roles in binding and catalysis. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the
most extensively studied 4-OT isozyme and the first characterized member of the tautomerase
superfamily, a group of structurally homologous proteins constructed from a β−α−β scaffold
with a catalytic Pro-1.
A search of five thermophilic bacterial genomes identified a coded amino acid sequence
annotated as a tautomerase-like protein in each that lacked Pro-1 (α-subunit). A nearby
sequence does contain Pro-1, but the sequence is not annotated as a tautomerase-like protein
(β-subunit). In order to characterize these proteins, two genes from Chloroflexus aurantiacus
J-10-fl were cloned, and the corresponding proteins expressed. Kinetic, biochemical, and Xray structural analysis show that the two expressed proteins form a functional heterohexamer
4-OT (hh4-OT), composed of three αβ dimers. Like the P. putida enzyme, the hh4-OT
requires the Pro-1 (from the β-subunit) and two arginines for the conversion of 2hydroxymuconate (1) to product (2), implicating an analogous mechanism.
A search of the databases with the α-subunit sequence of the hh4-OT uncovered an
enzyme designated as TomN, which reportedly converts 3 to 4 in a biosynthetic pathway for
the anti-tumor agent, tomaymycin. Our studies show that TomN, a homohexamer, catalyzes
the canonical 4-OT reaction using 1, although the Km value is slightly elevated. Again, Pro-1
and a pair of arginines are critical for the activity, suggesting an analogous mechanism. The
results raise questions about the functional assignment for TomN. The implications of the
new activities and structures for the superfamily are discussed.
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22nd Enzyme Mechanisms Conference
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January 2nd - 6th, 2011
O14
Kinetic, Mechanistic, and Structural Aspects of the cis-Prenyltransferase
Catalyzed Lipid Chain Elongation
Po-Huang Liang
Institute of Biological Chemistry & Genomic Research Center, Academia Sinica, Taipei,
Taiwan ROC
Isoprenoids comprise a family of more than 55,000 natural products with great structural
variety derived from 5-carbon isopentenyl diphosphate (IPP) and its isomer dimethylallyl
diphosphate (DMAPP). Allylic diphosphates such as farnesyl diphosphate (FPP) synthesized
from DMAPP and IPP serve as outlet points to a great variety of products. A group of
prenyltransferases catalyzing chain elongation of FPP to designated lengths by consecutive
condensation reactions with specific numbers of IPP are classified as cis- and trans-types
according to the stereochemistry of the double bonds formed by IPP condensation. The
complete kinetics of the multiple-step IPP condensation reactions by both types of enzymes
has been determined using steady-state and pre-steady-state approaches. As a result of solving
their crystal structures in conjunction with biochemical studies, more understanding into their
catalytic mechanisms, protein conformational changes and product chain-length determination
mechanisms are gained recently. Since these prenyltransferases play important functions,
potent inhibitors have been identified and their co-crystal structures have been solved for drug
development. In this talk, the current knowledge particularly on the cis-prenyltranferase
undecaprenyl diphosphate synthase which catalyzes consecutive condensation reactions of
FPP with 8 IPP to synthesize C55 lipid is summarized. Differences between the kinetics,
mechanisms, and structures of the cis- and trans-prenyltransferases are discussed.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O15
Genetic Code Reprogramming: Its Concept and Wide Applications
Hiroaki Suga
Department of Chemistry, School of Science, The University of Tokyo
The genetic code is the law of translation, where genetic information encoded in RNA is
translated to amino acid sequence. The code consists of tri-nucleotides, so-called codons,
assigning to particular amino acids. In cells or in ordinary cell-free translation systems
originating from prokaryotes or eukaryotes, the usage of amino acids is generally restricted to
20 proteinogenic (standard) kinds, and thus the expressed peptides are composed of only such
monomers. However, we recently devised a new means to reprogram the genetic code, which
allows us to express non-standard peptides containing multiple non-proteinogenic amino acids
in vitro. This lecture will describe the most recent development in the genetic code
reprogramming approach that enables us to express natural product-like non-standard
peptides. The technology involves (1) efficient macrocyclization of peptides, (2)
incorporation of non-standard amino acids, such as N-methyl amino acids, and (3) reliable
synthesis of libraries with the complexity of more than a trillion members. When the
technology is coupled with an in vitro display system, referred to as RaPID (Random Peptide
Integrated Discovery) system, the non-standard cyclic peptide libraries with a variety ring
sizes can be screened (selected) against various drug targets inexpensively, less laboriously,
and very rapidly.
References
Y. Goto, T. Katoh, H. Suga, Nature Protocols, in press.
T. Kawakami, A. Ohta, M. Ohuchi, H. Ashigai, H. Murakami, H. Suga “Diverse backbonecyclized peptides via codon reprogramming” Nature Chemical Biology 5, 888-890 (2009)
Y. Goto, K. Iwasaki, K. Torikai, H. Murakami, H. Suga “Ribosomal synthesis of
dehydrobutyrine- and methyllanthionine-containing peptides” Chemical Communication
3419-3421 (2009).
Y. Yamagishi, H. Ashigai, Y. Goto, H. Murakami, H. Suga “Ribosomal synthesis of cyclic
peptides with a fluorogenic oxidative coupling reaction” ChemBioChem 10, 1469-1472
(2009).
Y. Goto, H. Suga "Translation initiation with initiator tRNA charged with exotic peptides"
Journal of the American Chemical Society, 131, 5040-5041 (2009).
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O16
Engineering Translation with Non-natural Aminoacyl-tRNA Libraries
Nathan G. Uter, Brian Young, James E. Rozzelle, Juan Zhang, Junhao Yang,
& Christopher J. Murray
Sutro Biopharma, Inc. 310 Utah Ave, Suite 150, South San Francisco, CA 94073
Proteins and peptides containing non-coded amino acids represent an emerging class of drugs
that combine the power of protein engineering and design with unique chemistries. Sutro has
developed a scalable cell-free protein synthesis platform for efficient synthesis of proteins
containing site-specific non-natural amino acids (nnAAs). In contrast to in vivo methods of
incorporation that require orthogonal tRNA-synthetase pairs and are limited by cellular uptake
of nnAAs, we show rapid, high-yield production of designer non-natural aminoacyl-tRNAs
produced at large scale can be incorporated with extreme fidelity via ribosomal protein
synthesis. This approach greatly expands the functional chemical diversity space of nonnatural amino acids for engineering and design of novel therapeutic proteins at scale.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O17
Analysis of Non-enzymatic and Enzymatic RNA Phosphoryl Transfer
Reaction Mechanisms by Kinetic Isotope Effects
Mike Harris
RNA Center and Department of Biochemistry,
Case Western Reserve University School of Medicine,
Cleveland, OH
Phosphoryl transferase enzymes that act on DNA and RNA substrates are essential for cell
function. For many key examples active site residues have been pinpointed and specific
catalytic modes including acid/base catalysis and metal ion catalysis have been proposed.
Yet, the overall chemical mechanism and the interactions between enzyme and substrate that
stabilize the transition state remain ambiguous, even for well studied enzymes of this class.
Our goal is to integrate kinetic isotope effect (KIE) analyses of transition state charge
distribution with biochemical and computational methods (with Dr. Darrin York (Rutgers)) to
determine and compare the catalytic modes used in prototypical phosphoryl transfer enzymes
including ribozymes. We established characteristic nucleophile, leaving group and nonbridging oxygen KIEs for stepwise and concerted RNA strand cleavage reactions, and we are
determining how these values depend on interactions with acid/base and metal ion catalysts to
lay the foundation for studies of enzyme mechanism. Initial focus of enzyme studies is on
comparison of RNA 2’-O-transphosphorylation catalysts by the HDV ribozyme and by the
archetypical protein ribonuclease, RNase A. KIE results for catalysis by RNase A show that
the overall chemical mechanism for RNA cleavage is concerted with a late, product-like
transition state. Comparison of KIEs for RNase A at different pH reveals changes in leaving
group, but not nucleophile or non-bridging oxygen bonding in the TS that are likely to be
related to changes in active site protonation. To gain insight into mechanism and catalytic
interactions in the HDV ribozyme, we are determining KIEs for both Mg2+ and Mg2+independent reaction channels, and investigating the role of nucleobases in acid/base
catalysis. These experiments are designed to reveal transition charge distribution and establish
functional linkages between the scissile phosphodiester and active site components revealing
common and idiosyncratic features of catalysis by RNA and by protein enzymes.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O18
Structure/Function Studies of the Fosfomycin Resistance Enzyme FosB
Paul D. Cook and Richard N. Armstrong
Department of Biochemistry
Vanderbilt University Medical Center
Nashville, TN 37232
Fosfomycin is an antibiotic effective against a broad range of Gram-negative and Grampositive micro-organisms. Soon after its introduction into the clinical setting, however,
bacterial strains resistant to the drug appeared. Several mechanisms of resistance have since
been identified and characterized, including a group of enzymes that inactivate fosfomycin by
catalyzing a nucleophilic substitution on the drug. They are members of the vicinal oxygen
chelate superfamily of enzymes and are called FosA, FosB and FosX. FosA and FosX utilize
glutathione and water, respectively, as the nucleophile. FosB has been shown to utilize
cysteine, but with catalytic efficiencies lower than expected. Bacillithiol was recently
demonstrated to be a major thiol present in Gram-positive organisms, and it may be the
preferred nucleophile for FosB. In an effort to further characterize the mechanism of FosB
and to determine if it can utilize bacillithiol, we embarked on a combined structural and
functional study of this enzyme. We determined the 3-dimensional structure of FosB from
Staphylococcus aureus via X-ray crystallography. We are also in the process of producing
bacillithiol via enzymatic and chemical synthesis routes in collaboration with the Vanderbilt
Chemical Synthesis Core.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O19
Unmasking proton coupled electron transfer in ribonucleotide reductases
using unnatural amino acids
JoAnne Stubbe
Department of Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139
Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the
conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•)
cofactor to initiate catalysis. The initiation process requires long range proton coupled
electron transfer (PCET) over a 35 Å distance between the two subunits α and β by a specific
pathway (Y122W48Y356 within β to Y731Y730  C439 within α). The rate-limiting step in
nucleotide reduction is the conformational gating of the PCET process, masking the proposed
chemistry in the propagation pathway. 3-Nitrotyrosine (NO2Y) has recently been
incorporated site-specifically in place of Y122. We have recently shown that incubation of apoY122 NO2Y-β2 with Fe2+ and O2 generates a diferric-NO2Y• which has a half-life at 25ºC of
40 s. Sequential mixing experiments in which the cofactor is assembled to 1 NO2Y•/β2 and
then mixed with α2/CDP/ATP have been analyzed by stopped flow spectroscopy, rapid freeze
quench EPR spectroscopy and rapid chemical quench methods. These studies have for the
first time unmasked the conformational gating step. They reveal that the NO2Y• is reduced to
the nitrotyrosinate with biphasic kinetics (283 and 67 s-1), that dCDP is produced at >107 s-1,
and that a new Y• is produced at >100 s-1. PELDOR studies suggest that the new Y• is
located at 356 in β2.
A second unnatural amino acid, 3- aminotyrosine (NH2Y) has also been incorporated
site-specifically in place of Y356 ,Y731, and Y730. All three of these RNR mutants have been
examined in detail and can generate NH2Y• in a biphasic process with all substrate/effectorpairs (S/E) in a kinetically competent fashion. The slow rate (2.5 s-1) is the same for all S/E
pairs and is associated with conformational gating. All three RNRs are also active in
nucleotide reduction with rate constants of 0.3 to 0.7 s-1. These RNRs where radical
intermediates have been detected on the pathway, represent an excellent opportunity to study
PCET. The life-time of NH2Y• generated with α/β/S/E suggest that Kd for the subunit
interactions has increased relative to wt-subunit interactions. We have taken advantage of this
observation to obtain a model of the “active” RNR complex using negative staining electron
microscopy.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O20
Thrice Upon a Heme: redox reactivities of bacterial heme peroxidases
Sean J. Elliott,a Clinton F. Becker,a Oliver Einsle,b Katie E. Ellis,a Katherine E. Frato,a Alan
B. Hooper,c Gökçe Su Pulcua and Nicholas J. Watmoughd
a) Department of Chemistry, Boston University, 590 Comm. Ave., Boston, MA 02215
b) Institut für Biochemie, Albert-Ludwigs Universität Freiburg, Freiburg, Germany
c) Dept. of Biochemistry and Biophysics, Univ. of Minnesota, St. Paul, MN 55108
d) School of Biological Sciences, University of East Anglia, Norwich, U.K.
Cytochromes c are heme-bearing redox-active proteins that have at least one covalently
tethered protoporphyrin IX cofactor, attached to the protein via two Cys residues found in a
CXXCH sequence motif. We he have investigated the structure-function relationships of a
series of cytochrome c peroxidase enzymes from bacterial sources. Bacterial CCP enzymes
(bCCPs) possess two heme cofactors: a His/Met-ligated heme of high potential (H) and a
second peroxidatic heme of lower redox potential (L), and while all structurally characterized
bCCPs are strikingly similar in many ways, the enzymes from Pseudomonas aeruginosa
(Psa) and Nitrosomonas europaea (Ne) appear to operate through similar, yet distinct
mechanisms: while the Ne enzyme is fully active when both heme cofactors are oxidized, the
canonical Psa enzyme requires “reductive activation”, wherein H is poised at the reduced
state, suggesting fundamental differences in how the Ne and Psa enzymes store and
manipulate redox equivalents. In part, this difference can be understood in the resting state
(fully-oxidized) crystal structures that exist for the Ne and Psa enzymes, which reveal that a
single small His-bearing loop can bind to L in the case of the Psa enzyme, but is exists in an
‘open’ conformer in the Ne enzyme. Here, we have interrogated as a series of bCCPs using
direct electrochemistry, steady state kinetics and pre-steady-state reactivity. We have verified
that distinct differences in the redox-based mechanistic chemistry of the Ne-type and Psa-type
of enzyme can be monitored electrochemically: for example, rate-limiting electron-transfer
associated with the reduction of the ferryl intermediate of Ne CCP is in stark contrast to the
rate-limiting release of water from the active site of the Psa enzyme. Further studies of the
Geobacter and Shewanella CCP enzymes will show how specific amino acid side positions
contribute to the preference for one reactive manifold or another, and that a spectrum of
reactivity may be found amongst bacterial CCP enzymes.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O21
On the Mechanism of Radical SAM Methyltransferases RlmN and Cfr
Feng Yan,1,2 Jacqueline M. LaMarre,3 Alexander S. Mankin,3 Danica Galonić Fujimori1,2
1
Department of Cellular and Molecular Pharmacology and 2Department of Pharmaceutical
Chemistry, University of California San Francisco, 600 16th Street, San Francisco, California
94158, 3Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 S.
Ashland Ave., Chicago, Illinois 60607
RlmN and Cfr are Radical SAM methyltransferases that modify a single adenosine nucleotide
– A2503 – in 23S ribosomal RNA. This nucleotide is positioned within the peptidyl
transferase center of the ribosome, a site targeted by several antibiotic classes. Unlike other
RNA modifying methyltransferases, RlmN and Cfr carry out methylation of aromatic,
amidine carbons in the adenosine substrate to form 2,8-dimethylated product. This
modification of A2503 confers resistance to five classes of ribosome targeting antibiotics.
Recently, our lab carried out the first in vitro reconstitution of methylation catalyzed by
members of the Radical SAM superfamily, adding a new function to this diverse enzyme
class. We demonstrated that these enzymes use a novel addition-rearrangement mechanism to
achieve substrate methylation, thus expanding the repertoire of biological methyl transfer
reactions. This unusual mode of reactivity is enabled by the enzymes’ ability to use Sadenosyl-L-methionine (SAM) in two distinct roles: as a cofactor and a source of the
canonical 5’-deoxyadenosyl radical, and as a cosubstrate. Rather than providing the methyl
group, the cosubstrate SAM is a precursor to a methylene fragment incorporated into the
newly formed methyl group of the product. Additionally, by providing information on both
the timing of methylation and its substrate requirements, our findings have important
implications for the functional consequences of Cfr-mediated modification of ribosomal RNA
in acquisition of antibiotic resistance.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O22
Molecular Basis of Polyketide Cyclization
Shiou-Chuan Tsai
University of California, Irvine, CA
Ring formation is a common strategy to diversify natural products, and the molecular basis of
cyclization specificity is a vigorously pursued topic in natural product biosynthesis.
Polyketides are a class of natural products with highly diverse chemical structures and
pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase
(ARO/CYC) in bacterial aromatic polyketide synthase (PKS) and the PT domain in fungal
PKS, helps diversify polyketides. How the ARO/CYC and PT domain promotes highly
specific cyclization is not well understood. Herein we present the crystal structures of Tcm
ARO/CYC and the aflatoxin pksA PT domains. Although the two proteins share less than
15% of sequence identity, the crystal structures reveal a striking similarity in the overall fold,
substrate cavity and cyclization mechanism. The critical comparison allows a rationale behind
the vast cyclization diversity observed in polyketide natural products.
References
1. "Structure and function of an iterative polyketide synthase thioesterase domain
catalyzing Claisen cyclization in aflatoxin biosynthesis" Korman TP, Crawford JM,
Labonte JW, Vagstad AL, Wong J, Townsend CA, and Tsai SC. Proc. Natl. Acad. Sci.
USA. 2010, 107, 6246.
2. "Structural basis for biosynthetic programming of fungal aromatic polyketide
cyclization" Crawford JM, Korman TP, Labonte JW, Vagstad AL, Hill EA, KamariBidkorpeh O, Tsai SC, and Townsend CA. Nature, 2009, 461, 1139.
3. “Crystal Structure and Functional Analysis of Tetracenomycin ARO/CYC:
Implications for Cyclization Specificity of Aromatic Polyketides.” Ames BD, Korman
TP, Zhang W, Smith P, Vu T, Tang Y, Tsai SC. Proc. Natl. Acad. Sci. USA. 2008,
105, 5349.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O23
How Modular Polyketide Synthase Ketoreductases Have Evolved
to Set Different Combinations of Stereocenters
Jianting Zheng1, Shawn K. Piasecki2, Clint A. Taylor1, Josh F. Detelich1, Dionicio R. Siegel1,
Adrian T. Keatinge-Clay1,2
1
Department of Chemistry and Biochemistry
Institute of Cellular and Molecular Biology
The University of Texas at Austin
2
The ketoreductase (KR) domains of modular polyketide
synthases (PKSs) set the majority of stereocenters within
complex polyketides. Through an NADPH-coupled
reduction reaction a KR can set two stereocenters - the βcarbon that will bear the hydroxyl group and the substituentbearing α-carbon1. KRs isolated from their PKSs are
generally shown to act on a racemic mixture of diketide
substrate analogs to yield the products anticipated from the
natural reactions catalyzed by those KRs within their native
PKSs. Structural and functional data from A1-, A2-, B1-,
B2-, and C2-type KRs provide clues for how certain
stereochemical combinations can be faithfully generated2-4.
We propose that certain features of the KR active-site scaffold have evolved to enable the
synthesis of distinct combinations of stereocenters: 1) A Trp in A-type KRs and a Leu-Asp-Asp
in B-type KRs oppositely position the polyketide β-carbonyl in the active site, ultimately
resulting in the generation of an S or an R configuration at the β-carbon. 2) A general acid
enables A2, B2, and C2-type KRs to catalyze racemization to allow the epimerization of a
substituted α-carbon. 3) A residue three before the catalytic Tyr helps select the appropriate
epimer for reduction, leading to the generation of an S or an R configuration at the α-carbon.
References
1. Valenzano, C.R., Lawson, R.J., Chen, A.Y., Khosla, C., Cane, D.E. (2009). The biochemical
basis for stereochemical control in polyketide biosynthesis. J. Am. Chem. Soc. 131, 18501-11.
2. Zheng, J., Taylor, C.A., Piasecki, S.K., Keatinge-Clay, A.T. (2010). Structural and functional
analysis of A-type ketoreductases from the amphotericin modular polyketide synthase. Structure.
18, 913-22.
3. Keatinge-Clay, A.T. (2007). A tylosin ketoreductase reveals how chirality is determined in
polyketides. Chem. Biol.14, 898-908.
4. Keatinge-Clay, A.T., Stroud, R.M. (2006). The structure of a ketoreductase determines the
organization of the beta-carbon processing enzymes of modular polyketide synthases. Structure.
14, 737-48.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O24
Regulation of Histidine Kinases by H-NOX Domains
Lars Plate1, Mark A. Herzik1, Hans K. Carlson2, Mark S. Price1, W. Kaya Erbil2,
Kevan M. Shokat2,3, David E. Wemmer2, and Michael A. Marletta1,2,3
1
Department of Molecular and Cell Biology, 2Department of Chemistry, University of
California, Berkeley, Berkeley, CA 94720-3220 and 3Department of Cell and Molecular
Pharmacology, UCSF
It now clear that NO, a toxic, free radical, diatomic gas, plays a central role in cellular
function in eukaryotes (1). NO is both a signaling agent and acts in the host response to
infection. NO is synthesized by either constitutive isoforms of enzyme nitric oxide synthase
(NOS) or inducible isoforms of NOS. NOS catalyzes the conversion of arginine to citrulline
and NO. Using a low concentration of NO in signaling mitigates the toxicity problem but
places a difficult chemical requirement on the NO receptor, the soluble isoform of guanylate
cyclase (sGC). sGC contains a heme cofactor that traps NO, thereby activating the enzyme to
convert GTP to cGMP. The heme domain of sGC belongs the H-NOX (Heme-Nitric oxide /
OXygen) family of proteins with homologues in prokaryotes (2,3). Structural and
biochemical studies have provided a molecular explanation for selective NO trapping at low
concentrations (4). In addition, structural characterization has shown the heme in H-NOXs to
be highly distorted from planarity (5,6). Many of the H-NOX domains in prokaryotes are
contained in a predicted operon with a histidine kinase and often with other genes involved in
cellular signaling and gene regulation (2). For example, in Shewanella oneidensis the NObound form of the H-NOX selectively inhibits the constitutive autophosphorylation activity
kinase (7). Other evidence supports a NO sensor role for the S. oneidensis H-NOX signaling a
switch to low O2 growth conditions. Molecular characterization of H-NOX control over
histidine kinase activity has shown a role for relaxation of the distorted heme and associated
protein conformational changes that accompany ligand binding in kinase inhibition (8).
References
1. Marletta, M. A. (2004) Nitric Oxide Signaling, in Encyclopedia of Biological Chemistry (Lennarz, W. J.,
Lane, M.D., Ed.) pp 62-65, Elsevier, Amsterdam.
2.Iyer, L. M., Anantharaman, V., and Aravind, L. (2003) Ancient conserved domains shared by animal soluble
guanylyl cyclases and bacterial signaling proteins, BMC Genomics 4, 5.
3. Karow, D. S., Pan, D., Tran, R., Pellicena, P., Presley, A., Mathies, R. A., and Marletta, M. A. (2004)
Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and
Thermoanaerobacter tengcongensis, Biochemistry 43, 10203-10211.
4. Boon, E. M., Huang, S. H., Marletta, M.A. (2005) A molecular basis for NO selectivity in soluble guanylate
cyclase, Nature Chem. Biol. 1, 53-59.
5. Nioche, P., Berka, V., Vipond, J., Minton, N., Tsai, A.L., Raman, C.S. (2004) Femtomolar sensitivity of a NO
sensor from Clostridium botulinum, Science 306, 1550-1553.
6. Pellicena, P., Karow, D. S., Boon, E. M., Marletta, M. A., and Kuriyan, J. (2004) Crystal structure of an
oxygen-binding heme domain related to soluble guanylate cyclases, Proc Natl Acad Sci U S A 101, 1285412859.
7. Price, M. S., Chao, L. Y., and Marletta, M. A. (2007) Shewanella oneidensis MR-1 H-NOX regulation of a
histidine kinase by nitric oxide, Biochemistry 46, 13677-13683.
8. Erbil, W. K., Price, M. S., Wemmer, D. E., and Marletta, M. A. (2009) A structural basis for H-NOX signaling
in Shewanella oneidensis by trapping a histidine kinase inhibitory conformation, Proc Natl Acad Sci U S A
106, 19753-19760.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O25
Engineered Protein Methyltransferases for Bioorthogonal Target Labeling
Kabirul Islam,1 Weihong Zheng,1 Rui Wang,1 Gil Blum,1 Jamie McBean,1 Haiqiang (Bill)
Yu,2 Caitlin Sengelaub,1 Haiteng Deng2 and Minkui Luo1
1
Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center
2
Proteomics Resource Center, Rockefeller University, New York, NY 10065
Protein methyltransferases (PMTs) are a class of enzymes that deliver the sulfonium
methyl group from S-adenosyl-L-methionine (SAM) to specific lysine or arginine residues.
The sequence-specific posttranslational modification orchestrates numerous biological
processes. Aberrant PMT activities are frequently implicated in diseases including cancer.
Recent evidence also showed that PMTs function through methylating both histones and
nonhistone targets. Profiling methylation targets is an important but challenging task. To
address this challenge, we formulated a novel approach, which we termed as Bioorthogonal
Profiling of Protein Methylation (BPPM, Fig. 1), to elucidate the targets of designated PMTs
in the context of accurate disease models. As a key research module in the BPPM approach,
PMTs will be engineered to render the cofactor selectivity of SAM towards SAM analogues
and thus label their targets with distinct chemical groups in a bioorthogonal manner. Using
several cancer-relevant human PMTs as paradigms, we described our success in applying a
generalizable ‘bump-hole’ approach to evolve the PMTs to recognize a panel of SAM
analogues as cofactors. The structure-activity-relationship (SAR) analysis reveals the
importance of transition-state activation for the engineered PMTs to utilize the bulky SAM
analogues. The remarkable catalytic efficiency of several engineered PMTs makes the system
suitable to compete with native PMTs and SAM for target labeling. More importantly, the
clickable feature of several SAM analogues can further facilitate target enrichment prior to
MS analysis. Given the number of PMT variants and SAM analogues examined, and
conserved catalytic domains of PMTs, we envision that more such PMT/SAM variants can be
identified for PMT target profiling.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O26
The Enzymatic Activity of Sirtuins Uncovers Novel Protein
Posttranslational Modifications
Hening Lin
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
Sirtuins are a class of enzymes known as nicotinamide adenine dinucleotide (NAD)dependent deacetylases. Sirtuins regulate aging, transcription, and metabolism, and are
important targets for treating cancer, Parkinson’s disease, diabetes, and obesity. There are
seven sirtuins in humans, SIRT1-7. Four of them (SIRT4-7) have very little or no deacetylase
activity, which have caused many confusions and debates in the biological community. Our
work demonstrated that SIRT5, a mitochondrial sirtuin with very weak deacetylase activity,
catalyzes the hydrolysis of previously unknown acyl lysine modifications very efficiently. We
further identified proteins that are modified by these novel acyl groups from mammalian
mitochondria using mass spectrometry. This work demonstrated for the first time that sirtuins
can prefer acyl groups other than acetyl. SIRT5’s novel activity suggests that other sirtuins
with little or no deacetylase activity likely prefer other acyl groups too. This will greatly
facilitate the study of these sirtuins and possibly lead to the discovery of more novel protein
posttranslational modifications, some of which may be novel epigenetic modifications that
regulate transcription.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
O27
Painting the Cysteine Chapel: New Tools to Probe Oxidation Biology
Kate S. Carroll
The Scripps Research Institute, Department of Chemistry, Jupiter, FL 33458
The goal of our research is to understand molecular mechanisms of redox-based regulation of
protein function and the regulation by redox systems of complex physiological processes. To
achieve these goals, we develop and apply new technologies that cut across the traditional
boundaries of chemical and biological sciences.
Chemical Tools to Map Redox Signaling Pathways
Oxidative thiol modifications have emerged as a central mechanism for dynamic posttranslational regulation of all major protein classes and correlate with many disease states. We
focus on the development and application of chemoselective probes as tools to profile these
modifications in cell-based systems and to identify biomarkers of oxidative stress.
Thiolproteomic Technologies
New methods for comprehensive and quantitative analysis of the “thiol” proteome are being
developed and applied to the discovery of cellular signaling networks and regulatory
mechanisms that involve oxidation of protein cysteine residues.
Redox-Dependent Kinase and Phosphatase Inhibitors
Our lab is taking a rational design approach toward the development irreversible, smallmolecule inhibitors that target the oxidized forms of functionally important cysteine residues
in kinases and phosphatases.
Selected References
Depuydt, M.; Leonard, S. E.; Vertommen, D.; Denoncin, K.; Morsomme, P.; Wahni, K.;
Messens, J.; Carroll, K. S.; Collet, J. F., A periplasmic reducing system protects single
cysteine residues from oxidation. Science 2009, 326 (5956), 1109-11.
Seo, Y. H.; Carroll, K. S., Profiling protein thiol oxidation in tumor cells using sulfenic acidspecific antibodies. Proc Natl Acad Sci U S A 2009, 106 (38), 16163-8.
Leonard, S. E.; Reddie, K. G.; Carroll, K. S., Mining the thiol proteome for sulfenic acid
modifications reveals new targets for oxidation in cells. ACS Chem Biol 2009, 4 (9), 783-99.
Paulsen, C. E.; Carroll, K. S., Chemical dissection of an essential redox switch in yeast. Chem
Biol 2009, 16 (2), 217-25.
22nd Enzyme Mechanisms Conference
References
January 2nd - 6th, 2011
O28
Activity-based proteomics and its application for enzyme and inhibitor
discovery
Benjamin F. Cravatt
Department of Chemical Physiology, The Scripps Research Institute
Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms
universally possess a huge number of uncharacterized enzymes. The functional annotation of
enzymatic pathways thus represents a grand challenge for researchers in the genome era. To
address this problem, we have introduced chemical proteomic and metabolomic technologies
that globally profile enzyme activities in complex biological systems. These methods include
activity-based protein profiling (ABPP), which utilizes active site-directed chemical probes to
determine the functional state of large numbers of enzymes in native proteomes. In this
lecture, I will describe the integrated application of ABPP and complementary metabolomic
methods to discover and functionally annotate enzyme activities in mammalian systems,
including cancer and the nervous system. I will also present competitive ABPP platforms for
developing selective inhibitors for poorly characterized enzymes and discuss ongoing
challenges that face researchers interested in assigning protein function using
chemoproteomic methods.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P1
From mechanistic Enzymology to Biology:
Deciphering the Function of RubisCO-like Proteins
Tobias J. Erb, John A. Gerlt
Institute for Genomic Biology, University of Illinois at Urbana-Champaign
1206 W Gregory Drive, Urbana, IL 61801
D-Ribulose
1,5-bisphosphate carboxylase/oxygenase (RubisCO) is one of the most abundant
enzymes in the biosphere that catalyzes the carboxylation of ribulose-1,5-bisphophate to yield
two molecules 3-phosphoglycerate. Although it is the key enzyme in the Calvin cycle of CO2
fixation, RubisCO is considered not a perfect catalyst, because of a low turnover number and
non-productive side activities that compete with carboxylation, such as oxygenation,
β-elimination, and isomerization reactions. This catalytic promiscuity is an inherent property
of RubisCO, and it is not surprising that the active site architecture of RubisCO has been used
for the evolution of novel enzymatic activities within the RubisCO superfamily.
The RubisCO superfamily includes four distinct types of proteins. Whereas type I, II and III
RubisCOs are true CO2 fixing enzymes, type IV proteins lack residues essential for the
carboxylation reaction and are designated “RubisCO-like proteins” (RLP). These RLPs fall
into six divergent clades that all are expected to catalyze different reactions according to
active site residue differences and genomic context.
We recently assigned a novel function to the RubisCO superfamily, studying the mechanistic
diversity of the Rhodospirillum rubrum RLP in vitro. This RLP can use methylthioribulose-1phosphate as substrate to catalyze two subsequent enolization reactions. Further investigation
on the physiological significance of this new reaction in vivo was carried out using a
combined approach of RNA sequencing (RNAseq), knockout metabolomics, and cell extract
NMR. Our results led to the identification of a completely novel bacterial strategy to salvage
methionine from the dead end metabolite methylthioadenosine. This novel stratgey involves
the release of methanethiol (CH3-SH) from the carbon skeleton and is essentially different
from the canonical methionine salvage pathway.
In summary, our results demonstrate that at the dawn of postgenomic era, mechanistic
enzymology can serve as an important method for the functional assignment of unknown
genes. In combination with transcriptomic and metabolomic techniques, this represents a
powerful approach to mine microbial genomes for novel enzymatic reactions, novel metabolic
pathways and, therefore, novel biological principles.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P2
Lid dynamics in catalysis: A free energy rearrangement
Troy Johnson & Todd Holyoak
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center,
Kansas City, KS 66160.
Many studies have shown that dynamic motions of individual protein segments can play a role in
enzyme function. Recent structural studies on the gluconeogenic enzyme phosphoenolpyruvate
carboxykinase (PEPCK) have revealed a dynamic element in the form of a 10-residue omega-loop
domain that acts as an active site lid adapting an ordered, closed conformation upon the formation of
complexes that mimic the Michaelis and enolate intermediate states (Sullivan and Holyoak, 2008).
Based upon these structural studies we have previously proposed a model for the mechanism of
PEPCK catalysis in which the conformation of this mobile lid-domain is energetically coupled to
ligand binding resulting in the closed conformation of the lid, necessary for correct substrate
positioning, becoming more energetically favorable as ligands associate with the enzyme. The
increasing favorability of the formation of the closed lid state in progression from the holo enzyme
through the Michaelis and intermediate complexes suggests that this operates by a mechanism where
the entropic penalty of closing the lid is offset by the favorable Gibbs free energy of interaction
between enzyme and ligands as originally proposed by Jencks in his ‘Circe Effect’ paper (Jencks,
1975). Here we test our model by the introduction of point mutations designed to either increase the
entropic penalty for lid closure (A467G) or decrease/eliminate the transfer of energy from ligand
binding to stabilize the Ω-loop lid domain (E89A, E89D, E89Q). Kinetic studies demonstrate that all
four mutant PEPCKs are less active than Wt ranging from 75-90% decrease in kcat in the physiological
direction and 95-99% decrease in the reverse physiological direction. Further characterization reveals
that each mutant, during the conversion of oxaloacetate to phosphoenolpyruvate (PEP), does not
stabilize the lid closed conformation to the same degree as the Wt resulting in the reaction becoming
decoupled with the formation of pyruvate instead of PEP. This activity is not observed in the Wt
enzyme as this would lead to a futile metabolic cycle in vivo. To further substantiate our hypothesis
structural characterization was carried utilizing multiple complexes, which mimic the Michaelis and
enolate intermediate states. These complexes were the same ones previously solved for the Wt enzyme
in which the lid was observed to be in a 50-50 open/closed (Michaelis) or 100% closed (enolate)
conformation. Contrary to this observation, the mutant PEPCKs have a predominantly open
conformation (greater than 70%) in all complexes, which correlates with the kinetic data. Taken
together, the structural and kinetic characterization of these four mutant enzymes supports our model
for the role of the active site lid in catalytic cycle of PEPCK. The data further demonstrates that the
shift in the lowest energy conformation, between open and closed lid states, is a function of the free
energy balance between ligands binding to the enzyme and the entropic penalty for ordering of the
flexible Ω-loop lid domain.
1. Jencks, W. P. (1975). "Binding energy, specificity, and enzymic catalysis: the circe effect." Adv
Enzymol Relat Areas Mol Biol 43: 219-410.
2. Sullivan, S. M. and T. Holyoak (2008). "Enzymes with lid-gated active sites must operate by an
3.
induced fit mechanism instead of conformational selection." Proc Natl Acad Sci U S A 105(37): 1382934.
Johnson, T.A. and T. Holyoak (2010). “Increasing the Conformational Entropy of the Ω-Loop Lid
Domain in Phosphoenolpyruvate Carboxykinase Imapirs Catalysis and Decreases Catayltic Fidelity.”
Biochemistry 49: 5176-87.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P3
Establishing Structural Diversity in Ergot Alkaloids by an Old Yellow
Enzyme Homologue
Johnathan Z. Cheng†, Christine M. Coyle‡, Daniel G. Panaccione‡,
and Sarah E. O’Connor†
†
Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts Avenue,
Cambridge, Massachusetts 02139, and ‡West Virginia University, Division of Plant & Soil
Sciences, Morgantown, West Virginia 26506
Ergot alkaloids, secondary metabolites produced by filamentous fungi, elicit a diverse
array of pharmacological effects. Naturally isolated and semi-synthetic derivatives of these
alkaloids have been used for a wide range of medicinal purposes including the treatment of
migraines, parkinsonism, and tumor growth. Ergot alkaloids festuclavine 1 from Aspergillus
fumigatus and agroclavine 2 from Claviceps purpurea, derive from alternate enzymatic pathways
originating from the common biosynthetic precursor chanoclavine-I aldehyde 3.1 A mechanistic
understanding of ergot alkaloid ring formation may extend new insight into the biosynthesis of
other classes of natural products deriving from the intramolecular cyclization between indole and
prenyl moieties.
Here we describe how a homologue of Old Yellow Enzyme encoded in the Aspergillus
fumigatus ergot gene cluster catalyzes reduction of the α,β unsaturated alkene of chanoclavine-I
aldehyde 3 leading to the formation of festuclavine 1.2 In contrast, the Old Yellow Enzyme
homologue from Claviceps purpurea catalyzes the isomerization of the α,β unsaturated alkene of
chanoclavine-I aldehyde 3 to yield agroclavine 2. Further mutational analysis allows us to
propose a mechanistic rationale for how these two unique classes of ergot alkaloids are produced
by different fungi species.3
1. Schardl, C.L.; Panaccione, D.G.; Tudzynski, P., Ergot Alkaloids - Biology and Molecular
Biology. In The Alkaloids Cordell, G., Ed. Elsevier: 2006; Vol. 63, pp 45-86.
2. Cheng, J.Z.; Coyle, C.M.; Panaccione, D.G.; O'Connor, S.E., A Role for Old Yellow Enzyme
in Ergot Alkaloid Biosynthesis. J. Am. Chem. Soc. 2010, 132, (6), 1776-1777.
3. Cheng, J.Z.; Coyle, C.M.; Panaccione, D.G.; O'Connor, S.E., Controlling a Structural Branch
Point in Ergot Alkaloid Biosynthesis. J. Am. Chem. Soc. 2010, 132, (37), 12835-12837.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P4
Substituent Effects on the Reaction of β-Benzoylalanines with Pseudomonas
fluorescens Kynureninase
Sunil Kumar1, Vijay B. Gawandi1,2, Nicholas Capito1 and Robert S. Phillips1,3
1
2
Department of Chemistry, University of Georgia, Athens, GA 30602
Present address: The Department of Biochemistry and Biophysics, Texas A&M University,
College Station, TX 77843-2128
3
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA
30602
Kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic
cleavage of L-kynurenine to give L-alanine and anthranilic acid. β-Benzoyl-L-alanine, the
analogue of L-kynurenine lacking the aromatic amino group, was shown to a good substrate
for kynureninase from Pseudomonas fluorescens, and the rate-determining step changes from
release of the second product, L-Ala, to formation of the first product, benzoate (Gawandi, V.
B., et al., (2004) Biochemistry 43, 3230-3237). In this work, a series of aryl-substituted βbenzoyl-DL-alanines was synthesized and evaluated for substrate activity with kynureninase
from P. fluorescens. Hammett analysis of kcat and kcat/Km for 4-substituted β-benzoyl-DLalanines with electron withdrawing and electron donating substituents is nonlinear, with a
concave downward curvature. This suggests that there is a change in rate determining step for
benzoate formation with different substituents, from gem-diol formation for electron-donating
substituents to Cβ-Cγ bond cleavage for electron-withdrawing substituents. Rapid-scanning
stopped-flow kinetic experiments demonstrated that substituents have relatively minor effects
on formation of the quinonoid and 348 nm intermediates, but have a much greater effect on
the formation of the aldol product from reaction of benzaldehyde with the 348 nm
intermediate. Since there is a kinetic isotope effect on its formation from β,β-dideutero-β-(4trifluoromethybenzoyl)-DL-alanine, the 348 nm intermediate is proposed to be a vinylogous
amide derived from abortive β-deprotonation of the ketimine intermediate. These results
provide additional evidence for a gem-diol intermediate in the catalytic mechanism of
kynureninase.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P5
Analysis of the peroxiredoxin family: using active site structure and
sequence information for global classification and residue analysis.
Kimberly J. Nelson1, Laura Soito1, Stacy T. Knutson2,
Leslie B. Poole1*, and Jacquelyn S. Fetrow2*
1
Department of Biochemistry, Wake Forest University Health Sciences, Medical Center Blvd.,
Winston-Salem NC 27157
2
Departments of Physics and Computer Science, Wake Forest University,
Winston-Salem, NC 27109,
Peroxiredoxins (Prxs) are a widespread and highly expressed family of cysteine-based
peroxidases that react very rapidly with H2O2, organic peroxides, and peroxynitrite. Correct
subfamily classification has been problematic since Prx subfamilies are frequently not
correlated with phylogenetic distribution and diverge in their preferred reductant,
oligomerization state, and tendency towards overoxidation. We have developed a method that
uses the Deacon Active Site Profiler (DASP) tool to extract functional site profiles from
structurally characterized proteins, to computationally define subfamilies, and to identify new
Prx subfamily members from GenBank(nr). For the 58 literature-defined Prx test proteins, 57
were correctly assigned and none were assigned to the incorrect subfamily. The >3500
putative Prx sequences identified were then used to analyze residue conservation in the active
site of each Prx subfamily. Our results indicate that the existence and location of the resolving
cysteine varies in some subfamilies (e.g. Prx5) to a greater degree than previously appreciated
and that interactions at the A interface (common to Prx5, Tpx and higher order AhpC/Prx1
structures) are important for stabilization of the correct active site geometry. Interestingly, this
method also allows us to further divide the AhpC/Prx1 into four groups that are correlated
with functional characteristics. The DASP method provides more accurate subfamily
classification than PSI-BLAST for members of the Prx family. These results (1) have been
organized into a publically accessible, easily searchable, web-based database called the
PeroxiRedoxin Classification IndEX (PREX; http://www.csb.wfu.edu/prex/) containing
>3500 Prx protein sequences unambiguously classified into one of six distinct subfamilies (2).
This classification method can now be readily applied to other large protein families.
References
1. Nelson, K.J., Knutson, S.T., Soito, L., Klomsiri, C., Poole, L.B. and Fetrow, J.S. (2010)
Analysis of the peroxiredoxin family: using active site structure and sequence
information for global classification and residue analysis. Proteins, in press, DOI:
10.1002/prot.22936.
2. Soito, L., Williamson, C., Knutson, S.T., Fetrow, J.S., Poole, L.B. and Nelson, K.J. (2010)
PREX: PeroxiRedoxin classification indEX, a database of subfamily assignments
across the diverse peroxiredoxin family. Nucleic Acids Res., e-pub Oct 29, DOI:
10.1093/nar/gkq1060.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P6
A Chemico-Biological Rationale for the Use of Selenocysteine in a Redox
Active Enzyme: Focus on Mammalian Thioredoxin Reductase
Snider, Gregg W., Hondal, Robert J.
University of Vermont, Department of Biochemistry, Burlington, VT 05405
Mammalian thioredoxin reductase (mTR) is a homodimeric pyridine nucleotide disulfide
oxidoreductase that catalyzes the reduction of the catalytic disulfide bond of thioredoxin
(Trx). The enzyme contains both an N- and C-terminal dithiol/disulfide redox center with the
C-terminal redox center housed on a 16-amino acid extension containing a vicinal
selenosulfide bond as part of a tetrapeptide motif (Gly-Cys1-Sec2-Gly-OH). Mechanistically,
TR uses a hydride from NADPH to reduce a non-covalently bound FAD cofactor followed by
subsequent reductions of both N- and C-terminal redox centers through a dithiol/disulfide
interchange reaction. While the mammalian enzyme contains Sec, Cys-orthologues exist in a
number of higher eukaryotes and these Cys-enzymes catalyze an identical reaction without an
apparent loss of catalytic efficiency. The reduced selenolate (of Sec2 ) or reduced thiolate (of
Cys2) is thought to be responsible for attack on the disulfide bond of Trx. Selenium (Se) and
sulfur (S) are chalcogens which possess similar physical-chemical properties, with Se being
more nucleophilic and acidic than S (pKa of a selenol is ~5.2 vs. a typical thiol pKa of 8.5).
These two properties have long been cited as a type of “catalytic advantage” for Se relative to
S in enzymes. In contrast, the superior electrophilic character of Se has long been ignored by
biochemists. We assert that it is the superior electrophilicity of Se that is responsible for the
mechano-enzymatic advantage of Se in enzymes relative to S. Support for the hypothesis that
electrophilicity is important in the exchange reaction between N- and C-terminal redox
centers has been demonstrated by removing the C-terminal tail containing Se and then using
Se-containing surrogate substrates for the truncated enzyme. The truncated TR will reduce
small molecule Se-substrates fast, while S-homologues are reduced slowly. Other
electrophilic substrates such as ubiquinone are also substrates for the truncated TR. The
higher electrophilicity of Se has important biological consequences. For example RSeO2- can
be reduced very rapidly by thiols, while the reduction of RSO2- is extremely slow. This
suggested to us that Se-containing enzymes would resist irreversible inactivation by oxidation
by allowing the overoxidized Sec residue to recover more rapidly from oxidative stress. We
have shown that the Sec-containing TR is resistant to inactivation from 50 mM H2O2;
however, the Drosophila melanogaster Cys-TR was significantly inactivated under the same
conditions. Upon exposure to xanthine oxidase generated-superoxide (O2-), a one-electron
oxidant, Sec-TR remained active while Cys-TR activity was completely abolished. We posit
that the electrophilic character of the Se atom in the C-terminal redox center of Sec-TR is
critical for mechanistic recovery from both one- and two-electron oxidations. This provides a
rationale for the use of Sec in TR that is independent of any “catalytic advantage” that other
chemical properties of Se may confer to the enzyme relative to the use of S in Cys.
1. Snider, G., Grout, L., Ruggles, E.L., Hondal, R.J. (2010) Methaneseleninic acid is a
substrate for truncated mammalian thioredoxin reductase: Implications for the
catalytic mechanism and redox signalling. Biochemistry, In Press
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P7
The Mechano-Enzymatic Function of Selenocysteine In Thioredoxin
Reductase
Adam P. Lothrop, Robert J. Hondal
University of Vermont, Department of Biochemistry, Burlington VT 05405
Thioredoxin reductase (TR) is a homodimeric pyridine nucleotide disulfide oxidoreductase
that utilizes NADPH and two dithiol/disulfide redox centers to reduce its target substrate,
thioredoxin (Trx). Mammalian TRs replace cysteine (Cys) in the C-terminal redox center with
the rare amino acid selenocysteine (Sec). Sec is chemically more reactive than its sulfur
homolog, Cys, in a number of ways. However, Cys-containing TRs such as that of Drosophila
melanogaster (DmTR) can carry out efficient catalysis without the use of Sec. The key
question of this investigation is how Cys-TRs can compensate for the absence of the
chemically more reactive Sec residue. The traditional role ascribed to the Sec residue in TR is
as a superior nucleophile, which is thought necessary to attack the disulfide bond of Trx. In
contrast, we have recently presented evidence that the chemical property of Se that is
necessary for efficient enzymatic catalysis in TR is its superior electrophilicity. We have
termed this property the “mechano-enzymatic” function of Se. If Sec is required by
mammalian TRs for its superior ability to accept electrons relative to the S atom of Cys, then
how do Cys-containing TRs increase the electrophilicity of S relative to Se? Our hypothesis is
that Cys-TRs increase the electrophilicity of the C-terminal Cys1-Cys2 disulfide bond through
the use of catalytic geometry. This electrophilic activation mechanism requires the proper
positioning of a HisH+ residue to polarize the S1-S2 disulfide bond. This polarization renders
S2 (the equivalent of the Se atom in the mammalian enzyme) electron deficient, allowing for
efficient attack by the thiolate of the N-terminal redox center. To address our hypothesis, we
first disrupted the active-site geometry of DmTR by inserting one or two Ala residues in
between Cys1 and Cys2. The activity towards Trx decreased by 150-fold and 300-fold,
respectively, compared to WT DmTR. The activity of the Ala insertion mutants in DmTR
could be restored to near WT levels if Sec replaced the Cys2 residue. Insertion of Ala residues
lengthens the peptide backbone between Cys1 and Cys2. A second approach was to maintain
backbone length, but change the side chain length by substituting homoCys for either Cys1 or
Cys2. When homoCys substituted for Cys1, the activity of DmTR decreased 235-fold. In
contrast, when homoCys replaced Cys2 the activity only decreased 2-fold. We propose that
the reason that homoCys can replace Cys2, even with greatly altered catalytic geometry, is
that the close proximity of the S atom of homoCys2 in the mixed disulfide complex allows for
efficient attack by the thiolate of the N-terminal redox center, consistent with our electrophilic
activation mechanism. We could also use a truncated DmTR and replace the C-terminal redox
center with a peptide substrate that has a linear disulfide bond in contrast to the cyclic 8membered ring of the Cys1-Cys2 disulfide bond found in the enzyme. Analysis of the
structure-activity relationships of these peptide substrates show that a linear disulfide bond
could be an efficient substrate if the distance between S2 and the C-terminal carboxylate was
optimized at 6 atoms. This optimal size imparts rigidity to the disulfide bond allowing it to be
held in the correct catalytic position for reduction by the N-terminal thiolate, further
validating our electrophilic activation mechanism.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P8
Contribution of 5’ leader of the Precursor tRNA toward Recognition and
Catalysis by Bacterial RNase P
Wan Hsin Lim and Carol A. Fierke
Chemical Biology Program, Department of Chemistry, University of Michigan
930 North University Avenue, Ann Arbor, MI 48109
Ribonuclease P (RNase P) catalyzes the 5ʼ maturation of precursor-tRNA (pretRNA). Bacterial RNase P is composed of a catalytic RNA subunit (P RNA) and one
small protein subunit (P protein); both subunits contribute to substrate recognition.
Previous studies have suggested that RNase P recognizes mainly the tertiary fold of
the tRNA moiety. However, the leader of pre-tRNA interacts with the P protein
subunit. Furthermore, recent bioinformatics analyses of 5ʼ leader sequences in 160
bacterial genomes demonstrate species-dependent sequence preferences near the
pre-tRNA cleavage site, suggesting the formation of sequence-specific interactions
with the 5ʼ leader. Using Bacillus subtilis RNase P and pre-tRNAAsp, we demonstrate
that the affinity of RNase P for pre-tRNA is modulated by the sequence at the position
in the leader two and three nucleotides away from the cleavage site (N(-2) and N(-3)).
Substrates with adenosine and uracil at the N(-2) and N(-3) positions have the
tightest affinity for the Bacillus subtilis RNase P. Furthermore, we have identified a
non-conserved nucleotide, G319 at J18/2 of the P RNA, that forms a trans Watsoncrick-sugar-edge with the base at N(-2). In addition, alteration of the N(-3) nucleotide
from adenosine to cytosine modulates the Ca-dependence of pre-tRNA affinity,
suggesting that interactions with this nucleotide stabilize the active conformation of
RNase P.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P9
Glycosyltransferase Dynamics: Studying the Model System of MshA from
Corynebacterium glutamicum
Jacquelyn S. Turri and Patrick A. Frantom
Department of Chemistry, The University of Alabama Box 870336 Tuscaloosa, AL 35487
Glycosyltransferases (GTs) are enzymes that catalyze the transfer of a sugar moiety from an
activated donor molecule to a variety of acceptor molecules forming a glycosyl bond. Despite
the variety of substrates, GTs fall into two major structural superfamilies termed GT-A and
GT-B. The GT-B fold is comprised of two separate Rossman-like domains connected by a
flexible linker region. The catalytic site is located in the cleft between the two domains and is
completely formed after donor substrate binding initiates a conserved conformational change
in the enzyme. Our model system for investigating the importance of this conformational
change in catalysis is the retaining glycosyltransferase MshA from Corynebacterium
glutamicum (CgMshA). CgMshA forms a homodimer and is responsible for the first step in
mycothiol biosynthesis. The enzyme catalyzes the transfer of N-acteylglucosamine from
UDP-N-acetylglucosamine (UDP-GlcNAc) to 1-L-myo-inositol-1-phosphate (L-I1P) to form
the pseudo-disaccharide, GlcNAc-Ins-P. Previously reported crystal structures of CgMshA in
the presence and absence of the substrates show two distinct conformations of the enzyme,
with a 97° rotation of the C-terminal domain relative to the N-terminal domain occurring after
UDP-GlcNAc binding. To further investigate this movement Forster Resonance Energy
Transfer (FRET) was used to monitor the conformational dynamics of CgMshA in the
presence and absence of substrates. The two native cysteine residues in the enzyme structure
(C157 and C262) were mutated to alanine residues and one aspartate residue (D250) in the Cterminal domain of the enzyme was mutated to a cysteine residue to allow for FRET dye
conjugation. The labeled enzyme exhibits a ten-fold decrease in V/K(L-I1P) and a six-fold
decrease in V/K(UDP-GlcNAc) from native CgMshA values. There is also a three-fold decrease in
kcat values from 12 s-1 to 4 s-1 for native CgMshA and the dye-conjugated CgMshA variant
respectively. Ensemble FRET measurements were taken in the absence and presence of each
of the following substrates: UDP, UDP-GlcNAc, and L-I1P. A conformational change was
seen in the presence of UDP or UDP-GlcNAc, but not in the presence of L-I1P. This supports
the idea of contacts between the enzyme and UDP moiety initiating the conformational
change. The results indicate that the distance between C-terminal domains of the homodimer
changes from 72.7±0.4 Å to 65.2±0.4 Å from the open to closed conformations respectively.
This conflicts with the measurements from the open conformation crystal structure, which
could indicate that this conformation is a rare conformational transition state.
Figure 1: Overlay of open (PDB: 3C48
(Black)) and closed (PDB: 3C4V (Grey))
CgMshA crystal structures. A 97° rotation
can be seen from open to closed
conformations. The mutation site is
indicated on each monomer by the star.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P10
Glutamine-Dependent Asparagine Synthetase from Plasmodium berghei
Yong-Mo Ahn and Nigel G. J. Richards
Department of Chemistry, University of Florida, Box 117200, Gainesville, FL 32611.
Glutamine-dependent asparagine synthetase (ASNS) is an enzyme that catalyzes the
synthesis of asparagine from glutamine, ATP, and aspartate. In the crystal structure of
Escherichia coli ASNS, many aspects of the synthetase domain of the enzyme remain poorly
understood because there are several regions that are not observed. Therefore crystal structures of
asparagine synthetases from other organisms are needed to provide a better structural
understanding of ASNS if potent inhibitors are to be identified by rational discovery methods. In
this poster, we will describe recent work aimed at obtaining a crystal structure for the ASNS from
Plasmodium berghei.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P11
Biochemical and Biophysical Characterization of Human Pancreatic
Glucokinase
Mioara Larion and Brian G. Miller
Florida State University, Tallahassee, Fl, 32306
Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity for
the phosphorylation of glucose. To explain the unique allosteric properties glucokinase, two
models have been proposed. The mnemonic mechanism assumes the existence of one
thermodynamically favored enzyme conformation in the absence of substrates, while the
ligand-induced slow transition (LIST) model requires a preexistent equilibrium between two
enzyme species that interconvert with a rate constant slower than turnover. To investigate
whether either of these mechanisms is sufficient to describe glucokinase kinetic cooperativity,
we have employed a variety of biochemical and biophysical methods. Here we present the
results of genetic selection experiments that identified a variety of stimulatory substitutions
within the C-terminal α13 helix of glucokinase. We also demonstrate that deletion of the Cterminal α13 helix abolishes the cooperativity of the enzyme. These results establish a link
between the presence and identity of the primary amino acid sequence of helix α13 and the
allosteric properties of the enzyme. We also conducted a pre-steady state analysis of glucose
binding to glucokinase. Global fit analysis of these experimental glucose binding curves
revealed a minimal model for glucokinase kinetic cooperativity that includes two apo states
and four binary complexes. Finally, we collected heteronuclear NMR spectra of 15N-specific
labeled glucokinase in the absence and presence of glucose, which demonstrate that both
unliganded and glucose-bound enzyme sample multiple conformations. Taken together, these
data provide new insight into the mechanistic basis of cooperativity in monomeric human
glucokinase.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P12
pH Dependency of the Pericyclic Reactions Catalyzed by the
Isochorismate-Pyruvate Lyase from Pseudomonas aeruginosa
Jose Olucha, Qianyi Luo and Audrey L. Lamb
Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave, Lawrence, KS 66045
The isochorismate-pyruvate lyase (IPL) from Pseudomonas aeruginosa (PchB) breaks down
isochorismate into salicylate and pyruvate in a [1-5] sigmatropic hydrogen transfer from C2 to
C9 (kcat/Km = 4.1x104 M-1s-1).1,2 In addition, this enzyme catalyzes an adventitious chorismate
mutase (CM) [3,3] sigmatropic rearrangement of chorismate to prephenate (kcat/Km = 2.0x102
M-1s-1). There is an ongoing debate in the field over the relative contributions of formation of
a Near Attack Conformation (NAC) and Transition State Stabilization (TSS) to the molecular
mechanism of pericyclic reactions. The positive charge on the lysine 42 located on the active
site loop of PchB has previously been shown to be important for catalysis in both reactions.2
To determine the relative contributions of the NAC and TSS to the mechanisms of the lyase
and mutase reactions, we generated a lysine (pKa = 10.5) to histidine (pKa = 6.0) PchB
mutant, thereby giving this site a pH-dependent charge. Kinetic constants were determined
along a pH range from 3.5 to 9.5. For wild-type PchB, the kinetic parameters for both
activities remained constant from pH 4.5 to 9.5, whereas the catalytic efficiency decreased for
the K42H mutant at pH values corresponding to the deprotonation of the histidine sidechain.
However, the IPL and CM activities were not covariant: IPL activity was measurable at pH
values above 8 (see figure) but CM activity was not. These data suggest the positive charge at
position 42 is necessary for efficient catalysis in both IPL and CM activities in PchB. PchB
serves as a lyase without a positive charge stabilizing the transition state, suggesting that
formation of a NAC is sufficient for IPL catalysis. Chorismate mutase activity is not
observable above pH 8, suggesting that the positive charge at this site is necessary to stabilize
the developing negative charge of bond breakage.
References
1. DeClue, M.S., Baldridge, K.K., Kunzler, D.E., Kast, P. and Hilvert, D. (2005) J. Am.
Chem. Soc. 127, 15002-3.
2. Luo, Q., Olucha, J., and Lamb, A. L. (2009) Biochemistry 48, 5239-45.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P13
Directed Evolution of a Thermostable Quorum-Quenching Lactonase from
the Amidohydrolase Superfamily
Jeng Yeong Chow1,2, Bo Xue3, Robert C. Robinson3, and Wen Shan Yew1
1
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of
Singapore, Singapore; 2 NUS Graduate School for Integrative Sciences and Engineering,
Singapore; 3 Institute of Molecular and Cell Biology, Singapore.
A quorum-quenching lactonase identified from the thermophilic Geobacillus kaustophilus
was used as a template for directed evolution experiments. This enzyme belongs to the
amidohydrolase superfamily and is thought to be physiologically involved in the deactivation
of quorum-sensing pathways. More specifically, the enzyme catalyzes the hydrolysis of Nacyl-homoserine lactones (AHLs), a class of molecules often used by many gram-negative
pathogenic bacteria to mediate virulence.1 We obtained the structure of a catalytically-inactive
mutant (D266N) of this enzyme liganded with N-butyryl-L-homoserine lactone in the active
site, to a resolution of 1.6 Å. With the aid of a bioluminescence-based screen, we identified a
new mutant with enhanced catalytic efficiency and broadened substrate promiscuity. This new
mutant contains two mutations (E101N/R230I), located at the C-terminal ends of the 3rd and
7th β-strands, respectively. Characterization of this mutant revealed that it has a 72-fold
enhancement in kcat/KM towards 3-oxo-N-dodecanoyl-L-homoserine lactone. Interestingly,
although the purified wild-type and mutant enzymes were found to contain a mixture of zinc
and iron, they were colored differently at high concentrations. It had been proposed that such
coloration is mediated by the formation of a charge transfer complex between an iron cation
and Tyr99 within the active site of the enzyme.2 The observed enhancement in the lactonase
activity of our mutant could thus be attributed to favorable modulations of the enzyme active
site architecture.
References
1. Camilli, A., and Bassler, B. L. (2006) Science 311, 1113-1116
2. Xiang, D., Kolb, P., Fedorov, A., Meier, M., Federov, E., Nguyen, T., Sterner, R., Almo, S., Shoichet, B., and
Raushel, F. (2009) Biochemistry 48, 2237-2247
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P14
Studying the Substrate Tolerance of 4-Coumarate-CoA ligase from
Streptomyces Coelicolor
Maybelle K. Go, Lim Yan Ping, and Yew Wen Shan
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of
Singapore
4-Coumarate coenzyme A ligase (4CL) catalyzes the activation of 4-coumarate to 4coumarate-CoA thioester in a two-step reaction via an adenylate intermediate. It is a key
enzyme in the general phenylpropanoid metabolism that provides carboxyl-CoA thioester
precursors for a large variety of secondary metabolites such as flavonoids, lignin, and
polyketides. 4CL purified from various sources such as parsley, soybean, and potato were
found to catalyze the thioesterification of other derivatives of cinnamic acid other than 4coumarate (1,2).
4CL from Streptomyces coelicolor was expressed in Escherichia coli and purified to
homogeneity in a single step using Ni-chelating chromatography. Using the purified enzyme,
various derivatives of cinnamic acid, 3-phenylpropanoic acid, and benzoic acid were used to
screen 4CL’s catalytic activity towards the conversion of the various aromatic carboxylic
acids to their corresponding carboxyl-CoA thioesters. Aliphatic carboxylic acids were also
screened as possible substrates. This study found that 4CL was able to catalyze the CoA
thioesterification of various aliphatic and aromatic carboxylic acids, highlighting the potential
use of 4CL as a biosynthetic enzyme; the generated carboxyl-CoA thioesters can be used as
precursors in the biosynthesis of various secondary metabolites.
References
1.
Knobloch, K., and Hahlbrock, K. (1977), Archives of Biochemistry and Biophysics
184, 237-248.
2.
Beuerle, T., and Pichersky, E. (2002), Anal Biochem 302, 305-312.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P15
Synthesis and Screening a Haloacetamide Containing Library
Justin E. Jones,1,2 Jessica Slack,1,2 Corey P. Causey,2, and Paul R. Thompson1,2
1
2
Department of Chemistry, The Scripps Research Institute, Scripps Florida, 120 Scripps
Way, Jupiter, Florida 33458
Department of Chemistry & Biochemistry, University of South Carolina, 631 Sumter Street,
Columbia, SC 29208
Protein arginine deiminase (PAD) belongs to the guanidino-group modifying enzymes
superfamily. There are 5 mammalian enzymes PADs 1-4 and 6 that catalyze the conversion of
peptidyl-arginine to peptidyl-citrulline. Interestingly, the dysregulation of these enzymes are
involved in a number of diseases (1, 2). In particular, it has been shown that the dysregulation
of PAD2 as well as PAD4 are involved in a number of diseases including, rheumatoid
arthritis, multiple sclerosis, and cancer. Thus, inhibitors for these enzymes could represent
novel targets for the treatment of the associated diseases. Previously we have developed two
of the most potent mechanism based inactivators, F- and Cl-amidine of PAD4 (3, 4).
However, recent studies have determined that they are pan-PAD inhibitors (5). Herein, second
generation inactivators of PAD4 will be discussed. Furthermore, a potent and selective PAD4
inhibitor has been developed, TDFA, which is a mechanism based inactivator that displays
15-fold selectivity for PAD4 compared to PADs 1-3.
References
1.
Jones, J. E., Causey, C. P., Knuckley, B., Slack-Noyes, J. L., and Thompson, P. R.
(2009) Protein arginine deiminase 4 (PAD4): Current understanding and future
therapeutic potential, Curr Opin Drug Discov Devel 12, 616-627.
2.
Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., and Pruijn, G. J. (2003) PAD, a
growing family of citrullinating enzymes: genes, features and involvement in disease,
Bioessays 25, 1106-1118.
3.
Luo, Y., Arita, K., Bhatia, M., Knuckley, B., Lee, Y. H., Stallcup, M. R., Sato, M.,
and Thompson, P. R. (2006) Inhibitors and inactivators of protein arginine deiminase
4: functional and structural characterization, Biochemistry 45, 11727-11736.
4.
Luo, Y., Knuckley, B., Lee, Y. H., Stallcup, M. R., and Thompson, P. R. (2006) A
fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis,
and in vitro and in vivo evaluation, J Am Chem Soc 128, 1092-1093.
5.
Knuckley, B., Causey, C. P., Jones, J. E., Bhatia, M., Dreyton, C. J., Osborne, T. C.,
Takahara, H., and Thompson, P. R. (2010) Substrate specificity and kinetic studies of
PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase
3, Biochemistry 49, 4852-4863.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P16
Activity-Based Protein Profiling of Protein Arginine Methyltransferase 1
Obiamaka Obianyo,1,2 Corey P. Causey,2 Paul R. Thompson1,2
1
2
The Scripps Research Institute – Florida, Department of Chemistry, 130 Scripps Way
Jupiter, FL 33458
University of South Carolina, Department of Chemistry and Biochemistry, 631 Sumter St
Columbia, SC 29208
Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that
catalyze the mono- and di-methylation of peptidyl arginine residues. Although all PRMTs
produce mono-methyl arginine (MMA), type I PRMTs go on to form asymmetrically
dimethylated arginine (ADMA), while type II enzymes form symmetrically dimethylated
arginine (SDMA). The predominant member of the PRMT family is PRMT1, as it is
responsible for approximately 85% of the arginine methylation in mammalian cells (1). Since
PRMT1 exhibits type I activity, it is the primary producer of the competitive NOS inhibitor,
ADMA.
Recently, dysregulated PRMT1 activity has also been implicated in the
pathophysiology of breast cancer (2). Hence, potent inhibitors, which are highly selective for
this particular isozyme, could serve as therapeutics for heart disease and breast cancer.
However, the design of such inhibitors is impeded by a lack of information regarding this
enzyme’s mechanism of action and in vivo function. We have reported analyses of the
substrate specificity and kinetic mechanism of human PRMT1 using both an unmethylated
and a mono-methylated substrate peptide based on the N-terminus of histone H4. Our studies
have shown that PRMT1 preferentially methylates substrate peptides with positively-charged
residues distal to the methylation site (3). We have also determined that the enzyme
methylates its substrates in a partially-processive fashion, and utilizes a rapid equilibrium
random methylation mechanism with dead-end EAP and EBQ complexes (4). These results
have aided the design and synthesis of a potent PRMT1-selective inhibitor, denoted C21 (5).
Analyses of the inhibitor have demonstrated that C21 is able to covalently modify PRMT1,
suggesting its potential utility as an activity-based protein profiling probe to elucidate the in
vivo function of PRMT1 (6). Preliminary results obtained with fluorescent and biotinylated
derivatives of C21 will be described herein.
References:
1.
2.
3.
4.
5.
6.
Tang J, Frankel A, Cook RJ, Kim S, Paik WK, et al. 2000. J Biol Chem 275: 7723-30
Le Romancer M, Treilleux I, Leconte N, Robin-Lespinasse Y, Sentis S, et al. 2008.
Mol Cell 31: 212-21
Osborne TC, Obianyo O, Zhang X, Cheng X, Thompson PR. 2007. Biochemistry 46:
13370-81
Obianyo O, Osborne TC, Thompson PR. 2008. Biochemistry 47: 10420-7
Obianyo O, Causey CP, Osborne TC, Jones JE, Lee YH, et al. 2010. Chembiochem
11: 1219-23
Evans MJ, Cravatt BF. 2006. Chem Rev 106: 3279-301
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P17
Characterizing the Mechanism of PRMT1 Catalysis
Using Site Directed Mutagenesis
Heather Rust and Paul Thompson
Department of Chemistry, The Scripps Research Institute,
Scripps Florida, 120 Scripps Way, Jupiter, Florida, USA
Department of Chemistry and Biochemistry, University of South Carolina,
631 Sumter St., Columbia, South Carolina, USA
Post translational modifications of proteins play important roles in cellular functions.
For example, the methylation of arginine residues is involved in gene transcription, cell
growth, and proliferation. Arginine methylation is catalyzed by the protein arginine
methyltransferase (PRMT) family of enzymes, which transfer a methyl group from Sadenosylmethionine (SAM) to the guanidinium group of an arginine residue. This reaction
first produces monomethylated arginine (MMA) that can then be further methylated to
produce either asymmetrically dimethylated arginine (ADMA) or symmetrically dimethylated
arginine (SDMA). Type I PRMTs produce ADMA and Type II PRMTs produce SDMA. Of
the known PRMTs, PRMT1, which is a Type I enzyme, is the most abundant and well
characterized. Dysregulated activity of PRMT1 is believed to play a role in both heart disease
and cancer (1-3). The goal of our lab is to synthesize selective inhibitors for PRMT1 because
they may represent compounds that could be used to treat heart disease and cancer.
In order to design better inhibitors we must first thoroughly characterize the enzyme.
To investigate the proposed catalytic mechanism of PRMT1, active site mutants of highly
conserved residues, which are believed to play key roles in SAM recognition, substrate
binding, and catalysis (4), were made. The results show that Glu144 and Glu153, as well as
Arg 54, are critical for catalysis. Also, Tyr 39 and Met155 were found to play important roles
in SAM binding and processivity. Additionally, it was recently discovered that PRMT4,
which is also a Type I enzyme, is phosphorylated at a conserved serine residue and this
modification inhibits catalytic activity (5). However, we did not see the same result with a
mutation mimicking phosphorylation of PRMT1.
References
1.
2.
3.
4.
5.
Chen, X., Niroomand, R., Liu, Z., Zankla, A., Katus, H. A., Jahn, L., and Tiefenbacker, C. P. (2006) Expression of
nitric oxide related enzymes in coronary heart disease, Basic Res Cardiol 101, 346-353.
Le Romancer, M., Treilleux, I., Leconte, N., Robin-Lespinasse, Y., Sentis, S., Bouchekioua-Bouzaghou, K.,
Goddar, S., Gobert-Gosse, S., Corbo, L. (2008) Regulation of Estrogen Rapid Signaling through Arginine
Methylation by PRMT1, Molecular Cell 31, 212-221.
Osborne, T. C., Obianyo, O., Zhang, X., Cheng, X., and Thompson, P. R. (2007) Protein arginine methyltransferase
1: Positively charged residues in substrate peptides distal to the site of methylation are important for substrate
binding and catalysis, Biochemistry 46, 13370-13381.
Zhang, X., Cheng, X. (2003) Structure of the Predominant Protein Arginine Methyltransferase PRMT1 and
Analysis of Its Binding to Substrate Peptides, Structure 11, 509-520.
Feng, Q., He, B., Jung, S., Song, Y. Qin, J., Tsai, S.Y., Tsai, M., O’Malley, B.W. (2009) Biochemical Control of
CARM1 Enzymatic Activity by Phosphorylation, J. Biol. Chem. 284, 36167-36174.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P18
The Development and Use of Clickable Activity Based Protein Profiling
Agents for Protein Arginine Deiminase 4
Jessica L. Slack1, Corey P. Causey2, Yuan Luo2, Paul R. Thompson1
1
Department of Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps Way,
Jupiter, Florida 33458
2
The Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter
St., Columbia, South Carolina 29208
The Protein Arginine Deiminases (PAD), which catalyze the hydrolysis of peptidylarginine to form peptidyl-citrulline, are potential targets for the development of a Rheumatoid
Arthritis (RA) therapeutic, as well as other human diseases including colitis and cancer.
Additionally, these enzymes, and in particular PAD4, appear to play important roles in a
variety of cell signaling pathways including apoptosis, differentiation, and transcriptional
regulation. To better understand the factors that regulate in vivo PAD4 activity, we set out to
design and synthesize a series of activity-based protein profiling (ABPP) reagents that target
this enzyme. We successfully designed and synthesized six ABPPs including (i) FITC
conjugated F-amidine (FFA1 and 2) and Cl-amidine (FCA1 and 2), and (ii) biotin conjugated
F-amidine (BFA) and Cl-amidine (BCA). We have demonstrated the utility of these probes
for labeling PAD4 in cell, as well as for isolating PAD4 and PAD4 binding proteins. The
ability of PAD4 targeted ABPPs to enrich for endogenous PAD4 has enabled the
identification of several PTMs that PAD4 is subjected to, e.g. proteolytic processing,
ubiquitination, and autodeimination. These ABPPs in the future will enable the identification
of other post translational modifications that occur to PAD4 in vivo under a variety of
experimental conditions and should enable the identification of novel PAD4 interacting
proteins. Thus, these probes will undoubtedly prove to be powerful tools that can be used to
further dissect the factors controlling the dynamics of PAD4 expression, activity, and function
in normal and disease states.
References (1)Luo, Y.; Knuckley, B.; Bhatia, M.; Thompson, P. R. J Am Chem Soc 2006, 128, 14468. (2)Luo, Y.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.; Thompson, P. R. J Am Chem Soc 2006, 128, 1092. (3)Jones, J. E.; Causey, C. P.; Knuckley, B.; Slack, J. L.; Thompson, P. R. Curr Opin Drug Discov Devel 2009, 12, 616. 22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P19
Control of Mitochondrial Biogenesis by Mitochondrial Dynamics
Tianzheng Yu1, Yisang Yoon2
1
Alfaisal University College of Medicine, Riyadh, Kingdom of Saudi Arabia. 2Department of
Anesthesiology, University of Rochester School of Medicine and Dentistry, Rochester, New
York.
Mitochondrial shape change is mediated by fission and fusion of mitochondrial membranes.
Although physiological roles of the mitochondrial shape change is not fully understood, it has
been suggested that different shapes of mitochondria may reflect particular states of
mitochondrial activities. To understand the correlation between mitochondrial morphology
and function, we blocked mitochondrial fission in cells and examined its morphological and
functional consequences. The dominant-negative mitochondrial fission mutant DLP1-K38A
was used to inhibit mitochondrial fission. Cells overexpressing DLP1-K38A showed
elongated and entangled mitochondria.
Morphological quantification indicated that
mitochondrial fission decreased by 2-3 folds, consistent with the generation of overly
elongated mitochondria in these cells. Interestingly, however, the mitochondrial fusion
frequency also reduced to the similar extent when mitochondrial fission was inhibited,
suggesting the inter-dependence of fission and fusion processes. We found that inhibition of
mitochondrial fission slowed down cell growth and proliferation, suggesting an overall
decrease in cellular functions. In experiments testing the mitochondrial activity, we found
that cells overexpressing DLP1-K38A have a significantly higher mitochondrial membrane
potential compared to control cells. On the other hand, these cells showed decreased oxygen
consumption. Further experiments measuring the mitochondrial membrane potential in the
presence of the mitochondrial ATP synthase inhibitor oligomycine indicated that the proton
pumping activity of the electron transport chain in these cells decreased below the normal
activity. These cells also exhibited a lower reactive oxygen species level than control cells.
These results demonstrate that blocking mitochondrial fission causes a decreased activity of
the mitochondrial electron transport chain and a potential defect in dissipating the
electrochemical gradient across the inner mitochondrial membrane. Our results suggest that
changing mitochondrial morphology alters mitochondrial function, supporting the notion that
mitochondrial shape change is an active factor participating in controlling mitochondrial
activity.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P20
Kinetic Analysis of Malate Synthase from Mycobacterium tuberculosis
Christine Quartararo and John S. Blanchard
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY
Malate synthase is a 741 amino acid enzyme that catalyzes the Claisen condensation
of glyoxylate and acetyl-Coenzyme A (AcCoA) to generate malate in the glyoxylate shunt of
the citric acid cycle. The two enzymes of the shunt, isocitrate lyase, which converts isocitrate
to glyoxylate and succinate, and malate synthase are induced by low nutrient and hypoxic
conditions, which trigger the persistence phase in bacteria. The sequential activity of these
enzymes allows for the synthesis of carbohydrates and amino acids from AcCoA generated
from fatty acid oxidation. Humans lack the glyoxylate shunt, making this pathway an
attractive target for selective inhibition. Malate synthase is present in many pathogenic
bacteria and fungi including Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa, and
Candida albicans [1].
The malate synthase gene was cloned from Mycobacterium tuberculosis, and the
sequence-verified gene was cloned into the pET28a plasmid. Expression in E. coli yielded
soluble, N-terminally His6-tagged protein that was purified by Ni-NTA affinity
chromatography. An assay using chromogenic thiol detection and dithiodipridine was
developed, but suffered from loss of enzyme activity with increased pH. An active site
cysteine residue was suspected of reacting with the thiol reagent, and we mutated this residue
to a serine residue. The C619S mutant was as active as the wild type enzyme, and exhibited
linear kinetics. The enzyme requires a divalent metal ion, and preincubation with Mg2+ yields
enzyme with the highest activity. With this assay, we showed that the initial velocity pattern
was intersecting, indicating a sequential mechanism. The order of substrate binding has been
determined using product and dead-end inhibition. The pH dependence of kcat is bell-shaped,
decreasing below pH 6 as an enzyme catalytic base group exhibiting a pK value of 4.6 is
protonated and above pH 8 as an enzyme catalytic acid group exhibiting a pK value of 9.1 is
deprotonated. This defines a “window” where solvent kinetic isotope effects can be measured.
We have also synthesized trideutero-acetyl-CoA and will report on the magnitude of the
primary kinetic isotope effects on the reaction.
Reference
1.
Dunn, M.F., J.A. Ramirez-Trujillo, and I. Hernandez-Lucas, Major roles of isocitrate
lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 2009.
155(Pt 10): p. 3166-75.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P21
Hydroxymethylglutaryl-CoA lyase (HMGCL): Functional insights from
structures of HMGCL-Mg++-hydroxyglutaryl-CoA and HMGCL R41MMg++-HMG-CoA ternary complexes
Henry M. Miziorko1, Zhuji Fu2, Jennifer A. Runquist2, Jung-Ja P. Kim2
1
Division of Molecular Biology & Biochemistry, University of Missouri-Kansas City, Kansas
City MO 64110, and 2Biochemistry Department, Medical College of Wisconsin, Milwaukee
WI 53226.
HMGCL is crucial to ketogenesis and inherited human mutations result in potentially fatal
disease. Detailed understanding of the HMGCL reaction mechanism as well as the molecular
basis for human enzyme deficiency has been limited by the lack of structural information for
enzyme liganded to an acyl-CoA substrate or inhibitor. Soaking crystals of wild-type
HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA (HG-CoA) or of HMGCL
R41M with substrate HMG-CoA has supported determination of X-ray structures for liganded
HMGCL (2.4 Å) and liganded R41M (2.2 Å). Comparisons of these beta/alpha barrel
structures with those of unliganded HMGCL and R41M reveal substantial differences for
positioning of the flexible loop containing the conserved “signature” sequence of HMGCL. In
the ternary complex formed with substrate, Mg++, and R41M, loop residue C266 (implicated
in active site function by mechanistic and mutagenesis observations) is more closely
juxtaposed to the catalytic site than in the case of either unliganded enzyme or the complex of
wild-type enzyme with Mg++ and inhibitor HG-CoA. In the ternary complexes, the
physiologically active S-stereoisomer of substrate or of HG-CoA inhibitor is specifically
bound, in accord with the observation that oxygens from both the C3 hydroxyl and the C5
carboxyl groups are Mg++ ligands. Other Mg++ ligands are H233 and H235 imidazole
nitrogens, a D42 carboxyl oxygen, and an ordered water molecule. This water is positioned by
Mg++ and D42 within 3.3 Å of the C3 hydroxyl of bound acyl-CoA substrate/inhibitor and
may function as a proton shuttle. Results also indicate the interaction of R41 with the acylCoA’s C1 carbonyl oxygen, in agreement with the observed effects of R41 mutation on
reaction product enolization.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P22
A Heme-dependent Tyrosine Hydroxylase
Katherine L. Connor and Barbara Gerratana
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD
20742 USA
A tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster is
proposed to catalyze the ortho-hydroxylation of L-tyrosine to L-DOPA as the initial step of a
unique transformation to the hydropyrrole moiety of anthramycin (1). A multiple sequence
alignment of Orf13 with homologous enzymes LmbB2, TomI and SibU involved in the
biosynthesis of lincomycin A, tomaymycin and sibiromycin, respectively, provides little
information as to common motifs, folds and/or cofactor binding sites typically present in other
well characterized monophenol monooxygenases. We have identified heme b as a required
cofactor and observe maximal L-tyrosine conversion to L-DOPA in the presence of hydrogen
peroxide. This information provides initial classification of Orf13 as a heme peroxidase, a
known heme-dependent class of monophenol monooxygenases. Interestingly, we also observe
L-DOPA formation in the presence of L-ascorbate, a mild redox reagent, but with a turnover
number that is 40 fold less than with hydrogen peroxide alone. Little to no L-tyrosine
conversion is observed in the presence of stronger reducing agents. We are currently working
to optimize heme b incorporation for structural studies and to elucidate the ascorbate
dependent reaction.
References
1. Hu, Y., Phelan, V., Ntai, I., Farnet, C. M., Zazopoulos, E., and Bachmann, B. O. (2007)
Benzodiazepine Biosynthesis in Streptomyces refuineus, Chemistry & Biology 14, 691.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P23
The Structure of a C-3'-Methyltransferase Required for the Biosynthesis of DTetronitrose
Nathan A. Bruender,¶ James B. Thoden,¶ Manpreet Kaur,§ Marie K. Avey,§
and Hazel M. Holden¶
¶
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
§Edgewood Campus Middle School, Madison, Wisconsin 53711
Methylation is a common theme in biology where methyl groups are added to the carbons,
oxygens, sulfurs, or nitrogens of such molecules as proteins, nucleic acids, phospholipids, sugars,
and hormones, among others. Most of the enzymes responsible for
such methylation events require S-adenosylmethionine (SAM) as the
source of the methyl group. In recent years it has become
increasingly apparent that SAM-dependent enzymes play key roles
in the microbial biosynthesis of unusual di-, tri-, and tetradeoxy
sugars.
One such enzyme is a C-methyltransferase from
Micromonospora chalcea referred to as TcaB9. It is involved in the
production of D-tetronitrose (Figure 1), a tetradeoxy sugar found
attached to the antibacterial and antitumor agent tetrocarcin. The biosynthesis of this unusual
sugar requires 10 enzymes with TcaB9 catalyzing the fifth reaction, namely the transfer of a
methyl group from SAM to the 3'-carbon of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose.
For our X-ray analysis, two structures were determined to 1.5 Å resolution: one in which the
enzyme was crystallized in the presence of SAM and dTMP and the other with the protein
complexed to S-adenosylhomocysteine and its dTDP-linked sugar product. The overall fold of
the monomeric enzyme consists of three domains (Figure 2).
The N-terminal domain harbors the binding site for a zinc ion
ligated by four cysteines. The middle domain adopts the
canonical “SAM-binding” fold with a seven-stranded mixed βsheet. Likewise, the C-terminal domain contains a seven
stranded mixed β-sheet, and it appears to be related to the
middle domain by an approximate dyad, thus suggesting that
the present day version of TcaB9 arose via gene duplication.
Key residues involved in sugar binding and catalysis include
His 181, Tyr 222, Glu 224, and His 225.
The structure of TcaB9 represents the first protein model to
be
determined
by
Project
CRYSTAL
(http://www.projectcrystal.org/), an outreach effort established
in 2009 through funding by the NSF. One of the goals of Project CRYSTAL is to provide handson laboratory experience to middle school students. Details concerning both the structure of
TcaB9 and the efforts of Project CRYSTAL will be presented.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P24
Structural and Functional Studies of the Dehydrogenases Required for the
Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-D-mannuronic Acid
James B. Thoden and Hazel M. Holden
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
The lipopolysaccharide or LPS is the major structural component of the outer membrane
of Gram-negative bacteria. It is a complex glycoconjugate consisting of lipid A, the core
polysaccharide, and the O-antigen. Lipid A serves to anchor the sugar components of the LPS
to the bacterial cell membrane whereas the O-antigen, which extends farthest away from the
bacterium, contributes to the wide varieties of bacterial strains observed in nature. There is
growing evidence that O-antigens play important biological roles including, but not limited to,
virulence, effective colonization of host tissues, protection from phagocytosis and serummediated killing, and resistance to antimicrobial peptides.
O-antigens often contain some highly unusual dideoxy sugars including 2,3-diacetamido2,3-dideoxy-D-mannuronic acid, hereafter referred to as ManNAc3NAcA. This particular
sugar has been observed, for example, in the B-band O-antigen of Pseudomonas aeruginosa
O:3a,d, and in the A-band trisaccharide of the bacterium Bordetella pertussis, the causative
agent of whooping cough.
The dehydrogenases that are the topic of this investigation catalyze the second step in the
biosynthesis of ManNAc3NAcA, namely the oxidation of the C-3' hydroxyl group on the
UDP-linked sugar to a keto moiety and the reduction of NAD+ to NADH. Interestingly, there
are two distinct classes of these enzymes: those that require α-ketoglutarate for the
regeneration of NAD+, and those that simply release NADH. We will refer to these classes as
A and B, respectively. The Class A members are found, for example, in P. aeruginosa and
Thermus thermophilus, whereas the Class B members have been identified in B. pertussis and
Chromobacterium violaceum.
For the Class A family members, we have determined three high-resolution X-ray
structures: the enzyme with bound NAD(H), the enzyme in complex the NAD(H) and αketoglutarate, and the enzyme in complex with NAD(H) and its UDP-linked sugar substrate.
The Class A family members adopt highly unusual tetrameric quaternary structures with the
NAD(H) moieties positioned quite closely together. In addition, the UDP-linked sugars adopt
remarkably curved conformations when bound in the active site clefts. Contrary to what is
typically observed in other NAD-dependent enzymes, the NAD(H) binding pockets in the
Class A family members are shared among three subunits rather than contained within one.
The accommodation of α-ketoglutarate versus the UDP-linked sugar in these enzymes is
accomplished by simple flexibility in the protein region defined by Phe 256 to Leu 263.
For the Class B family members, we have determined the structures of the enzymes in
complex with NAD(H) and their UDP-linked sugars. The Class B enzymes adopt octameric
quaternary structures. Details concerning the structures and reactions mechanisms of these
dehydrogenases will be presented.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P25
Design of Non-covalent Small Molecule Inhibitors for Granzyme B
Mi-Sun Kim,† Dean A Heathcote, § Philip G Ashton-Rickardt,§ James P. Snyder†
†
Department of Chemistry, 1515 Dickey Drive, Emory University, Atlanta, GA 30322,
of Immunobiology, Division of Inflammation and Immunology, Department of
Medicine, Faculty of Medicine, Imperial College London, London W12 0NN
§Section
Over the last several decades, allogeneic bone marrow transplantation (BMT) has
emerged as an important therapeutic option for a number of malignant diseases. The
therapeutic potential of allogeneic BMT relies on the graft versus leukaemia (GVL) effect,
which eradicates residual malignant cells via immunological mechanisms. Unfortunately,
GVL outcomes are closely associated with graft-versus-host diseases (GVHD), the major
complication of allogeneic BMT, in which functional immune cells in the transplanted
marrow recognize the recipient as “foreign” and mount an immunologic attack. Furthermore,
recent work with granzyme B (GrB) deficient mice and humans has ascribed a critical role to
GrB in controlling GVL and GVHD. Therefore inhibition of GrB would be attractive for
therapeutic intervention to promote GVL and prevent GVHD.
GrB is a lymphocyte serine protease released by cytoplasmic granules within cytotoxic
T cells and natural killer cells to cause apoptosis. The enzyme is unique among mammalian
serine proteases for its strong requirement of Asp in the P1 position similar to caspases, a
family of cysteine proteases involved in apoptosis1. Small molecule serine protease inhibitors
naturally divide into two classes, covalent and non-covalent. However, the serine trap, an
electrophilic group in covalent blockers, possesses a reactive functional group and leads to a
lack of specificity because of the highly conserved Asp-His-Ser catalytic triad. Furthermore,
toxicity is a potential liability due to non-specific activity. Therefore, a key aim of this study
is to design non-covalent small molecule inhibitors by avoiding the electrophilic serine trap
and relying instead on ionic interactions, hydrogen bonding and hydrophobic contacts within
the binding groove. To explore this option, a computational model/screening approach has
been examined: (1) computational solvent mapping to identify “hot spots” in the active site
using FTMAP2; (2) virtual screening with three constraints (HB with Arg226, HP for S2 and
S4) based on the solvent mapping results3; (3) measure enzyme activity and selectivity; (4)
validate by modeling known covalent blockers4. a). As a result, novel classes of hGrB
inhibitors have been identified. In order to extend the pool of scaffolds, ‘lead hopping’ has
been carried out with ROCS to perform shape-based 3D database searches4. b). Moreover,
optimal and optimized structures will be synthesized and further refined.
References
1. a) Jennifer Rotonda et al. Chemistry & Biology. 2001, 8, 357-368; b) Yunyi Wei et al.
Chemistry & Biology. 2000, 7, 423-432; c) Robert V. Talanian et al. Journal of Medicinal
Chemistry. 2000, 43, 3351-3371
2. Ryan Brenke et al. Bioinformatics. 2009, 25, 621-627
3. Friesner, R. A. et al. Journal of Medicinal Chemistry. 2006, 49, 6177-6196
4. a) Christopher A. Willoughby et al. Bioorganic & Medicinal Chemistry Letters. 2002, 12,
2197-2200; b) ROCS: http://www.eyesopen.com/rocs
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P26
Long-range dynamic networks in the function and fidelity of poliovirus
RNA-dependent RNA polymerase
David D. Boehr, Xiaorong Yang, Jesse L. Welch
Department of Chemistry, Penn State University, University Park, PA
Positive-strand RNA viruses, such as poliovirus (PV), hepatitis C virus and SARS
coronavirus, represent existing and emerging threats to human health. One validated antiviral
target is the virus-encoded RNA-dependent RNA polymerase (RdRp). The error-frequency or
‘fidelity’ of PV RdRp has been previously shown to be critical to the pathogenesis of the
virus. For example, a remote-site mutation, Gly64Ser, in PV RdRp leads to a higher fidelity
polymerase that less readily incorporates the broad range antiviral compound, ribavirin, into
virus RNA, but viruses carrying the mutant polymerase are less capable of causing disease.
This suggests that RdRp fidelity is a potential target for antiviral strategies.
To better understand the molecular mechanisms of RdRp fidelity, we have begun
NMR-based studies to compare the solution structure and conformational dynamics of wildtype and fidelity mutant polymerases. Our studies suggest that there are long-range amino
acid networks that connect various functional parts of the enzyme, including regions
important for binding nucleotide and RNA, and for catalyzing phosphodiester bond
formation. Fidelity-altering mutations such as Gly64Ser result in structural and/or dynamic
changes to network amino acids, even to residues that are more than 30 angstroms from the
site of mutation. NMR structural analysis of ternary complexes bound with RNA and with
correct or incorrect nucleotide have given further insight into the fidelity-governing
conformational change step that precedes chemistry. There are structural changes in Gly64Ser
RdRp when bound with incorrect nucleotide that are not apparent in the wild-type enzyme.
Together, these studies provide much needed structural insight into the fidelity mechanisms of
PV RdRp, and they point towards novel antiviral strategies. We predict that mutations in the
amino acid networks that change fidelity may act as live attenuated vaccine strains.
Alternatively, small molecules that bind to and change the structure/dynamics of network
amino acids will change RdRp fidelity and act as novel antiviral compounds. In the future, we
will probe the protein conformational dynamics on the µs-ms timescale, the same timescale as
RdRp, to gauge the importance of protein fluctuations to polymerase function and fidelity.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P27
Kinetically Controlled Drug Resistance: How
Penicillium brevicompactum Survives
Xin Sun§, Bjarne Gram Hansen* and Lizbeth Hedstrom¶
Departments of §Biochemistry and ¶Biology, Brandeis University, Waltham, MA
*
Department of Systems Biology, Center for Microbial Biotechnology, Technical
University of Denmark, Denmark
Mycophenolic acid (MPA) is a billion-dollar immunosuppressant drug that acts by
inhibiting IMP dehydrogenase (IMPDH). MPA is a secondary metabolite produced by
several Penicillium strains and is toxic to many fungi species. How producer organisms avoid
self-intoxication remains unknown. The MPA biosynthetic cluster was recently cloned from
P. brevicompactum. Curiously, the cluster contains an additional IMPDH gene, suggesting
this IMPDH may confer P. brevicompactum’s resistance to its own drug.
IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of
NAD+ to NADH. The IMPDH reaction consists of two different reactions: oxidation and
hydrolysis. In the oxidation half of the reaction, the active site cysteine attacks the C-2
position of IMP, followed by a hydride transfer to NAD+, forming NADH and a covalent EXMP* intermediate. NADH is then released and E-XMP* is hydrolyzed in the second half
reaction to produce the XMP product. In most IMPDHs studied to date, hydride transfer is
fast and hydrolysis is rate-limiting. MPA inhibits IMPDH by trapping the E-XMP*
intermediate and preventing hydrolysis.
We have cloned, expressed and characterized both IMPDHs from P. brevicompactum
and show that both enzymes (A = not in MPA gene cluster, B = in cluster) are resistant to
MPA with IC50 = 425 nM and 27 µM for A and B, respectively. Furthermore, we show that
the remarkable resistance of the B enzyme is kinetically controlled: there is a change in the
mechanism such that hydride transfer is now the slow step and hydrolysis is fast. This
mechanism cleverly minimizes the build-up of the MPA-sensitive E-XMP* intermediate in
the steady-state, thereby rendering the enzyme insensitive to MPA. Most curious is the
finding that even at saturating MPA, the IMPDH B enzyme remains ~30% active.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P28
Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′phosphosulfate Reductase from Mycobacterium tuberculosis
Devayani P. Bhave1, Jiyoung A. Hong2, Michael Lee3, Wei Jiang4, Carsten Krebs3,4 and
Kate S. Carroll1,2,5
Chemical Biology Graduate Program1, Department of Chemistry2, University of Michigan,
Ann Arbor, Michigan, 48109-2216, Department of Biochemistry and Molecular Biology3 and
Department of Chemistry4, The Pennsylvania State University, University Park,
Pennsylvania, 16802, Department of Chemistry5, The Scripps Research Institute, Jupiter,
Florida, 33458
Mycobacterium tuberculosis adenosine 5'-phosphosulfate (APS) reductase (MtAPR) is an
iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for
the treatment of latent infection. The enzyme harbors a [4Fe-4S]2+ cluster that is coordinated
by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The ironsulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in
APR remains unknown. Progress in this area has been hampered by the failure to generate a
paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic
resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of
MtAPR in the [4Fe-4S]+ state. The EPR signal is rhombic and consists of two overlapping S =
 species. Substrate binding to MtAPR led to a marked increase in intensity and resolution of
the EPR signal, and to minor shifts in principle g values that were not observed among a panel
of substrate analogs, including adenosine 5′-diphosphate. Using site-directed mutagenesis, in
conjunction with kinetic and EPR studies, we have also identified an essential role for the
active site residue, Lys144 whose side chain interacts with both the iron-sulfur cluster and the
sulfate group of APS. These findings allude to a role for the iron-sulfur cluster in the catalytic
mechanism of APR.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P29
Dihydrofolate reductase: A correlation between the donor-acceptor
distance and its fluctuation to the catalyzed hydride transfer
Vanja Stojković, Laura Perissinoti, Stephen J. Benkovic, Amnon Kohen
Department of Chemistry, University of Iowa, Iowa City, IA 52242
Understanding the nature of the H-transfer and how the protein active site structure and dynamics
contribute to these reactions is of contemporary interest. Owing to the multidimensional and quantummechanical nature of a hydrogen transfer reaction, the rate of the transfer is sensitive to the protein’s
motions that modulate the height and the width of the reaction’s barrier. Subtle changes in the protein
structure, some of which take place on the time scale of the H-transfer step, might lead to more
efficient barrier penetration and H-tunneling. Here, we present kinetic data that together with
molecular dynamic simulations and crystallographic studies suggest that an active site mutation
modulates the nature of the hydride-transfer in the enzyme dihydrofolate reductase (DHFR; EC
1.5.1.3). DHFR is an enzyme that catalyzes a conversion of 7,8-dihydrofolate (DHF) to 5,6,7,8tetrahydrofolate through a simple chemical transformation (C-H→C transfer). The active site residue
that was modulated here is Ile 14, which is a conserved hydrophobic residue in contact with
nicotinamide ring of NADPH. We proposed that the role of this residue is to hold the nicotinamide
ring in the close proximity to pterin ring of DHF, and that active-site distance sampling is important
for the efficient catalysis. In order to examine this hypothesis, we measured the temperature
dependence of intrinsic KIEs and H-transfer rates of a series of active site mutants of Ile14 (Val1, Ala2,
Gly1), and compared it to those earlier measured for the wild type DHFR3. The wtDHFR exhibits
temperature independent intrinsic-KIEs, which indicates that its reaction coordinate is well organized
and where average donor-acceptor distance (DAD) is ideal for hydrogen tunneling. The temperature
dependence of the intrinsic-KIEs of these three mutants suggests a correlation between the average
DAD and its fluctuations and the size of the hydrophobic residue. That correlation indicated a poorly
reorganized reaction coordinate and an average DAD that is now too long for efficient tunneling, to be
associated with a smaller size of side-chain behind the H-donor. Molecular dynamic simulation
support kinetic studies and suggest that smaller size of residue 14 leads to new conformations with
broader distribution and longer average DADs (Figure 1).
Figure 1: Left: DHFR’s active site with I14, NADP+ and Folate highlighted.
Right: An MD generated histogram representation of the DAD distribution for each mutant.
References
1. Stojković, V.; Perissinoti, L.; Benkovic, S. J.; Kohen, A. PNAS in preparation.
2. Stojković, V.; Perissinoti, L.; Lee, J.; Benkovic, S.J..; Kohen, A. Chem. Comm. 2010, ASAP.
3. Sikorski, R.S.; Wang, L.; Markham, K. A.; Rajagopalan, P. T. R.; Benkovic, S. J.; Kohen, A. 2004
J. Am. Chem. Soc. 126, 4778-4779.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P30
Characterization of the Carboxybiotin Intermediate in Reactions of
Pyruvate Carboxylase.
Tonya N. Zeczycki1, Martin St. Maurice2, Sarawut Jitrapakdee3, John C. Wallace4, Paul V.
Attwood5, and W. W. Cleland1
1
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
53726,2Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin
53201,3Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand, 4School of Molecular and Biomedical Science, University of Adelaide, S.A., 5005,
Australia,5School of Biomedical, Biomolecular and Chemical Sciences, University of
Western Australia, Crawley WA, 6009 Australia.
The biotin prosthetic group acts as a mobile –CO2 carrier in the biotin-dependent
carboxylases, including acetyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate
carboxylase (PC) (1). The HCO3--dependent ATP-cleavage and subsequent carboxylation of
biotin at the N1-position occurs in the biotin carboxylase (BC) domain of PC, resulting in the
formation of the carboxybiotin intermediate. The covalently attached carboxybiotin then
swings to the carboxyl transfer domain where the –CO2 is transferred to pyruvate. Previous
studies have shown that the K1119Q PC mutant, a tetrameric apoenzyme lacking the
covalently attached biotin, still retains the ability to carboxylate free biotin in the presence of
HCO3- and MgATP (3). Carboxybiotin was enzymatically synthesized from 13C-enriched
HCO3- and characterized using 13C and 1H NMR (Scheme 1).
Scheme 1. Enzymatic synthesis of carboxybiotin.
The steady-state kinetics of the true reverse reaction of the BC domain, namely the formation
of MgATP from carboxybiotin, phosphate and MgADP, can now be further investigated. The
NMR methodologies presented can also be used to probe the mechanism of the oxamateinduced oxaloacetate decarboxylation reaction, the reverse reaction of the CT domain and
possibly determine the role of oxamate in the reaction.
(1) Perham, R. N. (2000) Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Ann. Rev. Biochem. 69, 961‐1004 (2) Jitrapakdee, S., St Maurice, M., Rayment, I., Cleland, W. W., Wallace, J. C., and Attwood, P. V. (2008)
Structure, mechanism and regulation of pyruvate carboxylase. Biochem. J. 413, 369-87.
(3) Adina-Zada, A., Jitrapakdee, S., Surinya, K. H., McIldowie, M. J., Piggott, M. J., Cleland, W. W., Wallace,
J. C., and Attwood, P. V. (2008) Insights into the mechanism and regulation of pyruvate carboxylase by
characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme. Int. J. of Biochem.
Cell Bio. 40, 1743-52.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P31
Mechanistic Studies of Methylphosphonate Synthase, a Non-Heme FeDependent Dioxygenase Linked to the Marine Carbon-Phosphorus Cycle
Heather A. Cooke, Wilfred A. van der Donk
Department of Chemistry, Institute for Genomic Biology, Howard Hughes Medical Institute,
University of Illinois at Urbana-Champaign, Urbana, IL 61801
Methylphosphonate synthase (MPnS) from Nitrosopumilus maritimus, an archeal marine
species, converts 2-hydroxyethyl phosphonate (2-HEP) to methyl phosphonate (MPn) and
either carbon dioxide or bicarbonate. MPn is then thought to be converted via reductive C-P
bond cleavage by ubiquitous C-P lyases to methane, a greenhouse gas, and phosphate, a
source of the essential nutrient phosphorus. MPnS is the only phosphonate biosynthetic
enzyme from an Archaeal species characterized to date and links carbon and phosphorus
metabolism in the ocean. MPnS is an unusual non-heme Fe-dependent dioxygenase. Its
closest characterized homolog is 2-hydroxyethylphosphonate dioxygenase (HEPD), which
acts on the same substrate yet affects a very different transformation, produces hydroxymethyl
phosphonate (HMP).1 Two mechanisms are postulated for MPnS. One involves two
successive hydroxylations of the substrate with an Fe(IV)-oxo intermediate, to produce MPn
and bicarbonate. The second mechanism occurs via hydroperoxylation of the substrate
followed by formation of a phosphonoacetate intermediate, and subsequent decarboxylation.
To elucidate the enzymatic mechanism, we explored the activity and kinetics of MPnS with 2HEP and HMP. Additionally, we examined other substrates under single-turnover conditions.
The products formed by these reactions gave insight into the mechanism. Furthermore, sitedirect mutagenesis was used to identify residues that are likely involved in Fe binding.
1
Cicchillo, R.M. et al. Nature 2009, 459, 871-874.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P32
Structural and Functional Significance of the Unstructured Loop Region of
Alkanesulfonate Monooxygenase
Jingyuan Xiong, Holly R Ellis
Department of Chemistry and Biochemistry
179 Chemistry Building Auburn University, Auburn, Alabama 36849
Bacteria are capable of utilizing alkanesulfonates as alternate sulfur sources when
inorganic sulfur is limiting in the environment. A two-component enzyme system comprised
of an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase
(SsuD) is expressed to generate sulfite from the desulfonation of alkanesulfonates. The threedimensional structure of SsuD exists as a TIM-barrel protein with an unstructured loop region
located above the active site [1]. In several TIM-barrel proteins, these loops are mobile and
are found to contribute to the structure of an enzyme active site or play a functional role both
in the binding of ligands and in enzyme catalysis [2]. The SsuD unstructured loop region is
proposed to possess similar flexibility as other TIM-barrel protein, with the loop region
closing over the active site with the binding of substrates. An interesting feature of SsuD is
that it utilizes FMNH2 as a substrate instead of a prosthetic group commonly found in many
flavoproteins. Since the FMNH2 substrate is transferred from SsuE to SsuD in aerobic
solutions, it would be easily oxidized by the oxygen in the bulk solvent if there is no adequate
protection provided by the enzyme. Therefore, the proposed loop-closing mechanism is
necessary for SsuD to prevent non-productive oxidation of FMNH2 following the transfer.
In order to evaluate the structural and functional significance of the SsuD unstructured
loop region, three variants containing a partial deletion of this region were constructed. All
three deletion variants were found to be catalytic inactive. Results from time-dependent
proteolysis experiments suggest that the length not the actual amino acid content of the loop
region is critical for the conformational change during substrate binding. Rapid kinetic
analyses were performed to investigate FMNH2 oxidation by the SsuD deletion variants under
single turn-over conditions. It was shown that the SsuD deletion variants were unable to
stabilize and protect the FMNH2 from autoxidation by O2. Visible circular dichroism spectra
showed the deletion variants were unable to alter the FMNH2 environment as observed for
wild-type SsuD, indicating SsuD fails to bind FMNH2 substrate without the intact
unstructured loop. The results of affinity chromatography experiments showed that the SsuD
deletion variant was still able to form static interactions with native SsuE, suggesting that the
SsuD loop region may not be involved in protein-protein interaction and FMNH2 transfer.
These results indicate that without the unstructured loop region the SsuD variants are unable
to facilitate FMNH2 binding, rendering the reactive FMNH2 substrate unstablized and
unprotected. In addition, the SsuD unstructured loop region may be responsible for triggering
crucial conformational changes during catalysis. Therefore, defects in this region of the SsuD
deletion variants may hinder necessary conformational changes and contribute to their loss of
activity. Overall, the results from these studies provide insight into the structural and
functional role of the SsuD unstructured loop in the desulfonation mechanism.
References
1. Eichhorn, E., Davey, C.A., Sargent, D.F., Leisinger, T., Richmond, T.J. (2002) Crystal
structure of Escherichia coli alkanesulfonate monooxygenase SsuD. J. Mol. Biol., 324,
457-468.
2. Farber, G.K., Petsko, G.A. (1990) The evolution of α/β barrel enzymes. trends Biochem.
Sci., 15, 228-234.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P33
Kinetic and Chemical Mechanisms of Homocitrate Synthase
from Thermus thermophilus
Vidya Prasanna Kumar and Paul F. Cook
Chemistry and Biochemistry, University of Oklahoma, Norman, OK
The α-aminoadipate pathway for lysine biosynthesis is nearly unique to higher fungi,
including human and plant pathogens and euglenoids. An exception is the thermophilic
bacterium Thermus thermophilus. Homocitrate synthase (HCS) catalyzes the first and
regulated step of the pathway, condensation of acetyl-CoA and α-ketoglutarate to give
homocitrate and CoA. Saccharomyces cerevisiae HCS (1) is stable only in presence of the
additives, guanidine hydrochloride, α-cyclodextrin, and (NH4)2SO4, which limits biochemical
and structural studies. As a result, the kinetic and chemical mechanism of the HCS from
Thermus thermophilus is being characterized. The Saccharomyces cerevisiae HCS is a Znmetalloenzyme, while the one from Thermus thermophilus requires Mn2+ or Mg2+ for activity.
With Mn2+ the kinetic mechanism of TtHCS is ordered with α-ketoglutarate (αKg) adding
prior to acetyl CoA (AcCoA), but with Mg2+ it is steady state random with the preferred order
the same as with Mn2+. The deuterium kinetic isotope effect measured with acetyl-d3 CoA is
unity at low AcCoA (Mg2+) for the pathway with αKg binding prior to AcCoA, but is about 2
for the pathway with AcCoA binding first. Data suggest presence of a slow conformational
change in the enzyme after binding of AcCoA to the enzyme concomitant with deprotonation
of the methyl of AcCoA. The solvent deuterium kinetic isotope effect is consistent with this
hypothesis and gives an isotope effect of 1.7. The proposed chemical mechanism is similar to
that proposed for Saccharomyces cerevisiae homocitrate synthase (ScHCS) with a Glu-His
dyad responsible for deprotonation of the methyl group of AcCoA (2). Lysine is a feedback
inhibitor and binds to the TtHCS active site, sharing some of the binding determinants of αKg
and giving competitive inhibition. Lysine exhibits negative cooperativity of binding,
indicating crosstalk between the two monomers of TtHCS. Binding to the first active site
reduces the affinity for binding of lysine to the second active site. Data are discussed in terms
of the mechanism of HCS and are compared to those obtained for the yeast enzyme.
This work was supported in part by funds from the Grayce B. Kerr endowment to the
University of Oklahoma to support the research of P.F.C.
References
1. Andi, B., West, A. H., and Cook, P. F. (2004) Stabilization and characterization of
histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. Arch.
Biochem. Biophys. 421, 243-254.
2. Qian, J., Khandogin, J., West, A. H., and Cook, P. F. (2008) Evidence for a catalytic
dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae.
Biochemistry 47, 6851-6858.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P34
Solvent Isotope Effects on the Desulfonation Mechanism of the TwoComponent Alkanesulfonate Monooxygenase System
John M. Robbins, Holly R. Ellis
The Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama, 36849
[email protected]
Two-component flavin-dependent monooxygenases are involved in various metabolic
and biosynthetic processes in microorganisms. Efforts to elucidate the details governing the
catalytic mechanisms of these systems continue to be an area of active investigation. The
alkanesulfonate monooxygenase enzyme is found in a diverse range of bacterial organisms
and utilizes free FMNH2 supplied by an independent NAD(P)H dependent FMN reductase
(SsuE) to alleviate periods of limited sulfur bioavailability. Catalysis by the monooxygenase
results in the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates to
yield free FMN, aldehyde, and metabolically available sulfite.1
Active site amino acid residues have been proposed to play a direct mechanistic role in
acid-base catalysis at specific steps in the reaction pathway. Sequence and structural analyses
of the monooxgenase enzyme were used to identify several conserved residues near the
proposed active site with the potential to contribute to catalytic function. Variants of these
amino acid residues were constructed and evaluated using different kinetic approaches
including chemical rescue experiments, single turnover kinetics, deuterium solvent isotope
effects, and pH dependence studies. The pH dependence of kcat indicated SsuD requires a
group with a pKa of 6.6 ± 0.1 to be deprotonated and a second group with a pKa of 9.5 ± 0.1 to
be protonated. The SIE on kcat was inverse and exhibited a dome-shaped proton inventory
curve. Results from single-turnover experiments show increased stability of the C4a(hydro)peroxyflavin intermediate in D2O, but that the overall rate of flavin oxidation by SsuD
monitored at 370 and 450 nm was not altered in the deuterated solvent. The results of the
present study indicated an active site arginine plays a substantial role in catalysis as any
mutation to this residue results in complete inactivation of the enzyme. These combined
results reveal critical catalytic features of this complex system.
References
1.
Eichhorn, E., Van der Ploeg, J. R., and Leisinger, T. (1999) Characterization of a two‐component alkanesulfonate monooxygenase from Escherichia coli, J. Biol. Chem. 274, 26639‐26646. 22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P35
Developing polyketide-based inhibitors targeting 3-dehydroquinate
dehydratase in the shikimate biosynthesis pathway.
Vivian W. N. Cheung§, Maybelle K. Go§, Robert C. Robinson‡ and Wen Shan Yew§
§
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of
Singapore, 8 Medical Drive, Singapore 117597, and the ‡Institute of Molecular and Cell
Biology, 61 Biopolis Drive, Singapore 138673.
Due to the emergence of drug-resistant pathogenic microbes, there is a pressing need
to develop new antibiotics to address the therapeutic needs of our generation. An example of
such bacterial resistance is that of the VREs, the Vancomycin-Resistant Enterococcus that
involves the Gram-positive commensal bacterium Enterococcus faecalis; this pathogen is
present in mammalian gastrointestinal tracts and has gained resistance to vancomycin, the
drug of “last resort”. We are targeting enzymes in the shikimate biosynthesis pathway as a
paradigm to develop the next generation of antibiotics. The shikimate biosynthesis pathway
is exclusive to microbes, plants and fungi, and thus is an attractive and logical target for
therapeutics aimed specifically at eliminating such pathogenic microbes. In this study, we are
interested in developing inhibitors that target 3-dehydroquinate dehydratase (DHQase, the
third enzyme in the shikimate pathway) from E. faecalis. We have determined the structure
of this enzyme, and have identified a number of polyketide inhibitors from compound library
screening. We are currently in the process of developing potential DHQase inhibitors using a
precursor-directed combinatorial biosynthesis route.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P36
Structural studies of MppR: An enzyme of unknown function from
enduracididine biosynthesis
Tyler Voegtline, Bilal H. Sayyed, Eric D. Lund, Nicholas R. Silvaggi
University of Wisconsin, Milwaukee, Department of Chemistry and Biochemistry,
3210 North Cramer Street, Milwaukee, WI 53211
Antibiotic-resistant pathogens are a serious and persistent threat to public health. The recent
isolation of methicillin-resistant Staphylococcus aureus (MRSA) with significant resistance to
vancomycin, the current last line of defense against this and other antibiotic-resistant
pathogens, is particularly ominous and underscores the need for new, effective antibiotics.
The nonribosomal peptide antibiotics mannopeptimycin and enduracidin are promising
candidates for development into chemotherapeutic agents for the treatment of MRSA,
vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae.
Mannopeptimycin is a lipoglycopeptide with a cyclic hexapeptide core of alternating D- and
L-amino acids. Enduracidin is a 17-residue cyclic lipopeptide. Both compounds contain the
rare, nonproteinogenic amino acids beta-hydroxy L-enduracididine or L-enduracididine,
respectively. In mannopeptimycin biosynthesis, L-enduracididine is produced from L-arginine
through the action of three enzymes, MppP, MppQ, and MppR. The details of this
transformation are currently unknown. The success of a semi-synthetic mannopeptimycin
analog, AC98-6446, has prompted much interest in producing additional derivatives of these
promising natural products. These efforts will be facilitated by a ready supply of
enduracididine and its analogs for use in combinatorial biosynthesis or semi-synthetic
approaches.
Herein we present the X-ray crystal structure of MppR (32.2 kDa) from Streptomyces
hygroscopicus at 1.6Å resolution (Rcryst=0.148, Rfree=0.170). The structure was determined by
SAD from a single data set collected from a SeMet-derivative crystal at LS-CAT beamline
21ID-D at the Advanced Photon Source. The overall fold of the protein is nearly identical to
acetoacetate decarboxylase (PDB ID 3BH2; SSM RMSD over 218 Cα atoms = 1.78Å). Given
the similarities between the tertiary structures of MppR and ADC, it is not surprising that
most of the active site residues are conserved, including the catalytic lysine (K156, MppR).
As in ADC, the pKa of amino group of this lysine side chain is reduced to approximately 6.5,
presumably by virtue of the highly apolar nature of the active site.
Despite the similarities, we have determined that MppR does not have acetoacetate
decarboxylase activity. Examination of the structure provides some explanation for this
finding. Several key ADC active site residues are not conserved in MppR. The arginine
residue that, in ADC, interacts with the carboxylate group of acetoacetate is replaced by a
tryptophan side chain in MppR. Likewise, the distal glutamate residue of ADC that has been
implicated in the decarboxylation mechanism is replaced by valine in MppR. Based on
structural and biochemical data, we propose a possible function for MppR in enduracididine
biosynthesis..
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P37
On the Analysis of Ligand Displacement Kinetics when a Probe is Rate
Contributing
Hidehisa Iwata1 and Mark S Hixon2
1
Discovery Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical
Company Ltd., Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan
2
Takeda San Diego, Inc., 10410 Science center Drive, San Diego, CA
Ligand binding assays operating through the displacement of an observable probe are often
employed for the determination of ligand binding constants. If the assay system is observed
continuously, it is possible to extract the microscopic rate constants kon and koff by progress curve
analysis when the probe is operating under rapid equilibrium conditions. When the probe
dissociation kinetics contribute to the overall displacement rates, conventional analytical
methods such as replot analysis produce anomalous results while global fitting to explicit 1-step
or 2-step ligand binding models fail to converge on well-fitting solutions. We present solutions
for ligand displacement systems when probe dissociation contributes to overall rate. By use of
equations describing 1-step or 2-step ligand slow binding but that also account for probe
contribution to rate, we solve the ligand displacement progress curves via global fitting and
extract the relevant microscopic rate constants for those systems.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P38
Discovery and design of inhibitors of dimethylarginine
dimethylaminohydrolase inhibitors by high throughput screening
Thomas Linsky, Corey Johnson, Yun Wang and Walter Fast
The University of Texas at Austin, 1 University Station A1900, Austin, TX 78712
From diabetes to drug addiction to hypertension and stroke to cancer, nitric oxide (NO) plays
a role in a growing number of diseases. In cancer specifically, overproduction of NO can lead
to increased growth and vascularity of numerous tumors. In humans, NO production is
regulated by the arginine analog asymmetric dimethylarginine. Dimethylarginine
dimethylarginase (DDAH) controls the level of this endogenous molecule by hydrolyzing it to
form citrulline and a corresponding alkylamine, and in turn increasing NO production. While
overexpression of DDAH leads to more NO, increased angiogenesis and faster-growing
tumors, inhibition of DDAH reduces NO levels and could show promise for slowing tumor
growth as well as treating other conditions where overproduction of NO is implicated. New
and potent inhibitors of DDAH are consequently of therapeutic interest.
To that end, robust assays for high throughput screening of the Pseudomonas aeruginosa and
human isoforms of DDAH were developed, and these enzymes were screened for in vitro
inhibition against over 6,000 compounds comprising both known drugs and chemically
diverse drug fragments. Of the 55 potential lead compounds identified, several chemically and
biologically relevant subsets were selected for further study. Despite nearly identical active
sites between the isoforms, several compounds exhibited preferential inhibition for one form
over the other. Another compound identified during screening is an anti-inflammatory drug
currently in phase-III clinical trials which was not previously known to interact with DDAH,
suggesting a possible alternative use. Inhibition by this compound is characterized in detail
and found to modify the catalytic Cys residue by formation of a selenosulfide bond. From the
remaining potential leads, three key structural motifs were identified for structure-activity
relationship studies, characterization and optimization. One subset, consisting of
halopyridines, reacts specifically with the catalytic Cys nucleophile of DDAH, but not with
free thiols. To our knowledge, these halopyridines have not been previously found to modify
any proteins. This inactivation is assisted by a conserved active-site Asp residue, which
appears to stabilize the cationic form of the pyridine ring. In addition to in vitro work,
mammalian cell studies with several of these compounds showed inhibition of DDAH,
demonstrating potential suitability as drug leads. Results and future paths for inhibitor
development will be presented.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P39
Substrate Specificity Amongst β-Ketoacyl-ACP Synthases
Janine Borgaro, Andrew Chang, Peter J. Tonge
Department of Chemistry, Stony Brook University, Stony Brook, NY, U.S.A., 11794-3400
Ketoacyl-ACP synthase (KAS) enzymes are components of the fatty acid biosynthesis
pathway which is a validated antibacterial drug target. The current research is focused on
substrate recognition by these enzymes. A key feature of the FASII system is that the
hydrophobic fatty acyl intermediates are shuttled from enzyme to enzyme attached to a
phosphopantetheine (PPant) prosthetic group on a small, highly acidic ACP, although many
will accept Coenzyme A (CoA). However, this is not true for some KAS enzymes. In order to
explore minimum requirements for catalysis, PPant and posttranslationally modified peptide
substrates have been synthesized and their ability to act as substrates for the KAS reaction
evaluated using steady state enzyme kinetics. Significantly it has been demonstrated that a
14-mer peptide derived from the ACP α2 recognition helix gives a kcat/Km value with the FabF
KAS enzyme that is virtually identical to that of the natural ACP-based substrate, indicating
that at least in this system the short peptide is an excellent replacement for the full-length
carrier. Finally, analysis of the structures of FabB and FabF KAS enzymes from E. coli
indicates that an arginine residue at the mouth of the pantetheine binding region plays a key
role in substrate binding. The importance of this residue in catalysis is currently being
evaluated by site directed mutagenesis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P40
Coenzyme-induced conformational changes in a flavin-dependent Nhydroxylating monooxygenase
Pablo Sobrado, Wyatt Chocklett, Elvira Romero, and Michelle Oppenheimer
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061
Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent N-hydroxylating enzyme
involved in the biosynthesis of ferrichrocin, a siderophore essential for virulence (1). Af SidA
catalyzes the NADPH- and O2-dependent conversion of N5-ornithine to N5-hydroxy
ornithine, which follows a sequential order kinetic mechanism (2). Formation of a C4aperoxyflavin has been observed, and is stabilized by NADPH and not by NADH.
Conformational changes in Af SidA upon coenzyme binding were assessed by examining the
CD spectra, its susceptibility to trypsin degradation, and the effect of titration with NAD(P)+.
The CD spectra show that binding of NADP+ causes significant changes in the secondary
structure of AfSidA compared to changes caused by NAD+ binding. Changes in the flavin
spectrum upon NADP+ binding are more pronounced than with NAD+. Limited proteolysis
studies in the absence of NADP+ show rapid degradation of Af SidA by trypsin. Addition of
NADP+ significantly decreased the rate of proteolysis by trypsin. Addition of ornithine
decreases the rate of degradation only in the presence of NADP+. Kinetic isotope effects were
measured under steady-sate conditions. With NADPD, a Dkcat value of 3.2 ± 0.2 and a
D
kcat/KM value of 2.7 ± 0.2 were measured. Using NADD, the Dkcat and Dkcat/KM values were
2.0 ± 0.1 and 2.5 ± 0.3, respectively. Together, the kinetic, spectroscopic, and biochemical
data suggest that in these flavin-dependent monooxygenases NADP(H) plays a dual role in
catalysis, flavin reduction and stabilization of flavin intermediates.
References
1. Hissen, A. H., Wan, A. N., Warwas, M. L., Pinto, L. J., and Moore, M. M. (2005) The Aspergillus
fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for
virulence, Infect. Immun. 73, 5493-5503
2. Chocklett, W.S, Sobrado, P. (2010) Aspergillus fumigatus SidA is a specific ornithine hydroxylase
with bound flavin cofactor. Biochemistry. 49. 6777-83
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P41
Histidine 252 in Adenosine 5’-phosphosulfate Reductase Is Required for
Substrate Binding
Jiyoung A. Hong1 and Kate S. Carroll2
1
2
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109
Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, 33458
Tuberculosis (TB) is still the leading cause of death from a bacterial infectious disease.
Adenosine 5’-phosphosulfate reductase (APR) catalyzes the first committed step in bacterial
sulfate reduction and is essential for mycobacterial survival in the latent phase of TB
infection. The sulfonucleotide undergoes a nucleophilic attack by the catalytic cysteine
(Cys249) to form an enzyme-S-sulfocysteine (E-Cys-Sγ–SO3–) intermediate and the enzyme
adopts a closed conformation with the C-terminal tail over the active site. A conserved
residue in the C-terminal tail, His252 is suggested to play a key role in the APR mechanism.
Substitution of His252 with alanine affects APS reduction, as indicated by a 130-fold
decrease in kcat/Km, 50-fold increase in Kd value and 100-fold increase in Km, kcat when
compared to the corresponding kinetic parameters for wild-type APR. However, His252Ala
mutant is exhibited no apparent difference in kmax.
Since the pKa of the catalytic cysteine (Cys249) is not perturbed, by the His252Ala
mutation, there is no evidence that His252 acts as a catalytic base to deprotonate this key
residue (pKa = 6). pH dependence studies and the binding affinity (Kd) of a substrate
analogue, 5’-phosphoaristeromycin indicate that His252 interacts with the ribose oxygen (O4)
and α-phosphate in the substrate. Thus, these data suggest that His252 participates directly in
substrate binding not catalysis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P42
Characterization and purification of 3-deoxy-D-manno-oct-2-ulosonic acid
hydroxylase, KdoO from Burkholderia ambifaria.
Hak Suk Chung, Christian R. H. Raetz
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 USA
KdoO from Burkholderia ambifaria and Yersinia pestis is a novel Kdo 3-hydroxylase that
converts the outer 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) unit of Kdo2-lipid A to Dglycero-D-talo-oct-2-ulosonic acid (Ko) in an O2, Fe2+, and α-ketoglutarate dependent
manner. KdoO is the first example of a sequenced deoxy-sugar hydroxylase and contains the
putative iron-binding motif, HXDXn>40H. Homologues of KdoO are found exclusively in
Gram-negative bacteria, including the human pathogens Burkholderia mallei, Yersinia pestis,
Klebsiella pneumoniae, Legionella longbeachae, and Coxiella burnetii, as well as the plant
pathogen Ralstonia solanacearum. KdoO from Burkholderia ambifaria (BaKdoO) is a
membrane-associated protein that is solublized by either detergent or high salt. Purified
BaKdoO-His6 utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate but not Kdo-lipid IVA or
heptosyl-Kdo2-lipid A in vitro. Therefore KdoO may function after the Kdo-transferase KdtA
but before the heptosyl-transferase WaaC in vivo. Activity of BaKdoO-His6 was stimulated
in the presence of Triton X-100 and displayed optimal activity at pH 7.0 - 7.5. Activity
measurements for alanine mutants of the potential iron-binding site residues: H140XD142,
H202, H213, H219, and H254 in BaKdoO suggested that H140XD142 and H219/H254 play an
important role in Fe2+ binding. These experiments set the stage for structural and
mechanistic. This study is supported by NIH GM51796.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P43
4-Oxalocrotonate Decarboxylase and Vinylpyruvate Hydratase from
Leptothrix cholodnii SP-6 May Act on a Chlorinated Form of the Substrate:
Analysis and Quantitation
Stephanie Taylor1, Christian P. Whitman2
1
Department of Chemistry and Biochemistry, The University of Texas at Austin 78712-1071
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin
78712-1071
2
4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH), form a
complex in the catechol meta-fission pathway, and convert 2-oxo-3-hexendioate to 2-oxo-4hydroxypentanoate1. Although the Pseudomonas putida mt-2 enzymes have been extensively
studied, many structural and mechanistic questions remain unanswered. Recently, 4OD/VPH homologues have been identified in Methylibium petroleiphilum, Leptothrix
cholodnii SP-6, and Comamonas sp. Strain CNB-1. Observations reported for the
Comamonas enzymes suggest that these homologs may act on a chlorinated form of the
substrate2, but this is not conclusive. Methlyibium petroleiphilum is also of interest because
this strain may degrade certain gasoline additives. In order to better understand how the
complex works on the substituted substrates, the genes for the enzymes from Leptothrix
cholodnii SP-6 are being cloned, the proteins expressed and purified, and their properties
delineated. In addition, a coupled assay is required to quantify the reaction products and
possible intermediates. Coupling the 4-OD/VPH homologs to 4-hydroxy-2-ketovalerate
aldolase and lactate dehydrogenase would allow the reaction to be monitored by the decrease
in the absorbance of NADH. The coupled assay requires that the gene for the aldolase be
cloned, the protein expressed and purified, and its properties delineated. The cloning of the
aldolase is complete, and expression and purification are being optimized. To date, the genes
for the homologue of 4OD/VPH from Leptothrix cholodnii SP-6 have been cloned
individually, but, like the enzymes from Pseudomonas putida mt-2, are inactive and
insoluble1. To solve this, they will be cloned dicistronically to enhance coexpression.
References
1. Stanley, T. M., Johnson, W. H., Jr., Burks, E. A., Whitman, C. P., Hwang, C.-C., and Cook,
P. F. (2000) Expression and stereochemical and isotope effect studies of active 4oxalocrotonate decarboxylase, Biochemistry 39, 718-726
2. Wu, J., Jiang, C., Wang, B., Ma, Y., Liu, Z., and Liu, S. (2006) Novel Partial Reductive
Pathway for 4-Chloronitrobenzene and Nitrobenzene Degradation in Comamonas sp. Strain
CNB-1, Applied and Environmental Microbiology 72, 1759-1756
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P44
Identification of the allosteric response element(s) in the active site of
mammalian pyruvate kinase isozymes
James Urness, Cody Timmons, Kelly Thuet, and Aron W. Fenton
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center,
Kansas City, KS
All four mammalian pyruvate kinase isozymes catalyze the same enzymatic reaction, the
transfer of phosphate from phoshpoenolpyruvate (PEP) to ADP to produce pyruvate and ATP.
However, allosteric regulatory properties are unique to each isozyme. As an example, the
affinity of M1-PYK (expressed in brain and muscle) for PEP is reduced upon binding
phenylalanine. PEP affinity of the liver isozyme (L-PYK) is reduced in the presence of
alanine, but increased upon addition of fructose-1,6-bisphosphate (Fru-1,6-BP). The
commonality in these allosteric systems is the influence on PEP affinity. Here we explore
whether all forms of allostery (activating or inhibiting) in multiple isozymes (rabbit M1-PYK
or human L-PYK) influence the same active site/substrate interaction(s). To initiate this
study, the allosteric properties of a series of PEP analogues were characterized. Titration of
intrinsic protein fluorescence of M1-PYK was used to determine effector binding over a
concentration range of each substrate analogue. The phosphate moiety of PEP interacts with
the active site K+ ion; removal of the phosphate moiety from the ligand reduces binding
affinity, but does not prevent the allosteric response. Likewise, modifications at most
positions in the PEP ligand do not prevent the allosteric response. The one position that we
have not fully probed is the carboxyl group of the substrate; to-date, synthesized
phosphorylated analogues with modifications of the carboxyl moiety have not been
sufficiently stable to allow analysis. By elimination, we speculate that the interaction between
the carboxyl moiety of PEP with the active site divalent cation is likely the interaction that
plays a role in allostery. The same analogue series will give insights into the region of the
active site in L-PYK that respond to alanine and Fru-1,6-BP. Consistent with the potential
allosteric role of the divalent/carboxyl interaction, replacement of Mg2+ with Mn2+ removes
Fru-1,6-BP activation in L-PYK, and greatly reduces the inhibition caused by alanine in the
same isozyme.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P45
Characterization of substrate binding loop of 3α-hydroxysteroid
dehydrogenase/carbonyl reductase
Chi-Ching Hwang*, Yu-Mei Su and Hsin-Wei Chen
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical
University, Kaohsiung, Taiwan
The substrate-binding loop in the short-chain dehydrogenases/reductase (SDR)
appears to be one of the most variable parts in the structure. The substrate binding loop
(T188-G213) in the structure of the 3α-hydroxysteroid dehydrogenase/carbonyl reductase
(3α-HSD/CR) located between the βF sheet and the αG helix appears disordered. The study
in kinetic isotope effects suggests the release of NADH is the rate limiting step in the reaction
of androsterone with NAD+ catalyzed by 3α-HSD/CR1. To enhance the enzymatic reaction
rate, we try to increase the rate for dissociation of NADH by engineering the flexible substrate
binding loop.
We compared the structure of apoenzyme with NAD+ bound binary complex and
observed the decrease in distance by 0.19 Å between S114 and P185, and found an additional
part of substrate binding loop in the binary complex, including the residues T188, E189, T190
and P191. In addition, the binding of NADH with S114A mutant induces the conformational
change observed in the CD and fluorescence spectrum2. In this study we investigate the roles
of P185 and T188 in the conformational changes of the substrate binding loop and enzyme
catalysis through site-directed mutagenesis, spectrophotometric analysis of conformational
changes by via of circular dichroism and fluorescence spectroscopy, and steady-state kinetics.
Mutants of P185A, P185G, P185W, T188A, T188S, T188W and double mutants of
T188W/W173F and P185W/W173F 3α-HSD/CRs are generated to probe its role in
conformation changes and catalysis. Mutation on the P185 causes the change of secondary
structure while mutation on T188 shows a similar CD spectrum with WT enzyme. An
increase in the catalytic constants kcat, Michaelis constant Km for androsterone and inhibition
constants KiNAD was observed for mutants of P185A, P185G and T188A. These results
indicate P185 and T188 in the substrate binding loop are important in binding with nucleotide
cofactor and androsterone. Mutants of P185A, P185G and T188A cause the increase the
dissociation of nucleotide cofactor, thereby increase the rate of releasing product. These data
may help to unravel mechanisms of the flexible substrate binding loop of 3α-hydroxysteroid
dehydrogenase/carbonyl reductase.
References
1.Chang, Y.-H., Chuang, L.-Y., and Hwang, C.-C. (2007) "Mechanism of Proton Transfer in
the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni" J.
Biol. Chem. 282, 34306-34314.
2.Chang, Y.-H., Huang, T.-J., Chuang, L.-Y., and Hwang, C.-C. (2009) "Role of S114 in the
NADH-induced conformational change and catalysis of 3α-hydroxysteroid
dehydrogenase/carbonyl reductase from Comamonas testosteroni" Biochim. Biophys. Acta
1794, 1459–1466.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P46
Promotion of Oligomeric DNA Polymerase Complex Formation Dictates
Alternative Enzymatic Activities in Archaea
Michael A. Trakselis, Zhongfeng Zuo, Hsiang-Kai Lin, Robert Bauer
University of Pittsburgh, Department of Chemistry
DNA polymerases are essential enzymes in all domains of life for both DNA
replication and repair. We find that two B-family DNA replication polymerases (Dpo1
and Dpo3) as well as the Y-family DNA repair polymerase (Dpo4) from Sulfolobus
solfataricus physically associate with themselves and the resulting complex is
stabilized in the presence of DNA. Various biochemical and biophysical techniques
were used to establish that a single Dpo1 DNA polymerase binds to DNA with higher
affinity followed by the cooperative binding of two additional molecules at higher
concentrations. On the other hand, Dpo3 and Dpo4, bind as homodimers to DNA.
We have determined that in all cases these are specific polymerase-polymerase
interactions around DNA. These oligomeric DNA polymerase complexes have unique
thermodynamic binding parameters. Specifically, the heat capacity of binding (ΔCp)
for Dpo1 and Dpo4 vary dramatically with temperature due to changes in the coupled
equilibria of the individual systems. Interestingly, the unusual trimeric Dpo1 DNA
polymerase complex has an increased kinetic rate of synthesis, extremely long
processivity, DNA annealing, and terminal transferase activities. Although the
monomeric Dpo1 polymerase is fully functional, the trimeric polymerase affords
additional activities that result in efficient activities at high temperatures in DNA
replication or repair. Unlike for Dpo1 or Dpo4, dimeric Dpo3 formation is absolutely
required for polymerase activity. Taken together, these results suggest that formation
of an oligomeric DNA polymerase complex is able to process DNA substrates more
efficiently than the monomeric form, especially at higher temperatures. This has
implications for coordinated DNA replication on the leading and lagging strands as
well as serves as a model for polymerase exchange at the replication fork.
References
1. Mikheikin A.L., Lin, H-K., Mehti, P., Jen-Jacobson, L., and Trakselis, M.A. (2009) A
trimeric DNA polymerase complex increases the native replication processivity. Nuc. Acids
Res., 37, 7194-205.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P47
MshB – A Cambialistic Metallohydrolase?
M. Hernick, X. Huang, E. Kocabas
Biochemistry Department, Virginia Tech, Blacksburg, VA 24061
Mycothiol (MSH) is the primary reducing agent used by mycobacteria, and is also involved in
drug/toxin detoxification. MshB is a metal-dependent deacetylase that catalyzes the
hydrolysis of N-acetylglucosamine-inositol (GlcNAc-Ins) to form glucosamine-inositol
(GlcN-Ins) and acetate, a key step in the biosynthesis of MSH. Consequently, MshB is a
target for the development of antibiotics for the treatment of tuberculosis. Zinc was reported
to be the physiological cofactor for MshB; however, the experiments that led to this
conclusion were carried out under conditions that favor zinc incorporation over Fe(II).
Therefore, we have subsequently examined the metal requirements of MshB. Results from
our studies demonstrate that MshB is a mononuclear metalloenzyme with the activity of Fe2+MshB > Zn2+-MshB, suggesting that Fe2+ may be a physiologically relevant cofactor for
MshB. We used pull-down experiments to rapidly purify MshB under anaerobic and aerobic
conditions and identify the cofactor(s) that purify with MshB under various conditions.
Results from these experiments indicate that MshB preferentially contains a bound Fe2+ when
purified under anaerobic conditions, while MshB copurifies with Fe2+ or Zn2+ as the metal
content of the growth medium is varied under aerobic conditions. Together, these results
suggest that MshB may be a cambialistic metallohydrolase that utilizes Fe(II) or Zn(II) under
different environmental conditions. Finally, we have started to probe the factors that
determine metal incorporation into MshB. Results from these studies indicate that MshB has
a higher affinity for Zn2+ compared to Fe2+, while both metal ions have similar dissociation
rate constants from MshB. These findings may suggest that metal incorporation is guided by
the association rate constant of the metal ions to MshB. Additional experiments are underway
to determine the identity of the cofactor bound under biologically relevant conditions and
identify the factors that dictate cofactor preferences of this enzyme.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P48
Probing the mechanism of DpgC, a unique cofactor and metal-free
dioxygenase involved in the biosynthesis of vancomycin
Heather Condurso1, Elisha Fielding2, Paul Widboom3, Steve Bruner4
1
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, 2Scripps Institution of
Oceanography, UC San Diego, La Jolla, CA 92037, 3Department of Materials Science and
Engineering, MIT, Cambridge, MA 02139, 4Department of Chemistry, University of Florida,
Gainesville, FL 32611
DpgC is a unique cofactor and metal-free dioxygenase involved in the biosynthesis of the
potent antibiotic vancomycin. A co-crystal structure of DpgC with a substrate mimic bound
was solved in 2007 and shed some light upon the activity of this enzyme. The crystal
structure revealed an oxyanion hole, which is a hallmark of the crotonase superfamily of
which DpgC is a member. Given this information we have proposed a radical mechanism,
which fits with the biochemical data. Currently, we are using synthetic chemistry to develop
mechanistic probes to validate our proposed mechanism.
Figure 1: Proposed mechanism of DpgC
The substrate of DpgC is a coenzyme A (CoA) tethered thioester. The first substrate mimic
synthesized replaced the thioester with the more stable amide and was found to be an inhibitor
of the enzyme, preventing even the first step of enolization. Several other derivatives are
being investigated that should provide further evidence for each step of the mechanism.
Figure 2: Structures of mechanistic probes in development. The remainder of the CoA
portion has been omitted for clarity.
References
1. Widboom, P.F., et al. Nature (2007) 447, 342.
2. Fielding, E.N., et al. Biochemistry (2007) 46, 13994.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P49
Functional Promiscuity of Rhodospirillum rubrum RuBisCO for RuBisCOLike Protein Reactions
Benjamin Warlick* and John A. Gerlt*†
University of Illinois, Urbana-Champaign Department of Biochemistry* and Institute of
Genomic Biology.† Institute of Genomic Biology MC-195, 1206 W. Gregory Dr., Urbana, IL,
61801
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant
protein on Earth. It has immense ecological significance, because it catalyzes the fixation of
atmospheric carbon dioxide using ribulose-1,5-bisphosphate to produce two molecules of 3phosphoglycerate. Recently, a fourth class of RuBisCOs was discovered that does not
catalyze the fixation of carbon dioxide, these are called “RuBisCO-like proteins” (RLPs).
Evidence presented by Ashida et al.1 has suggested that Rhodospirillum rubrum RuBisCO can
catalyze the tautomerization of 2,3-diketo-5-methylthiopentane-1-phosphate to 2-hydroxy,3keto,5-methylthiopentene-1-phosphate as performed by the RLP from Bacillus subtilis.
Curiously, R. rubrum has both an RLP and a RuBisCO. The RLP of R. rubrum catalyzes a
1,3 proton shift using 5-methylthioribulose-1-phosphate to generate a mixture of 1methylthioxylulose-5-phosphate and 1-methylthioribulose-5-P2. We investigated, in vitro, the
ability of R. rubrum RuBisCO to perform the B. subtilis RLP reaction and its own RLP
reaction. We present several lines of evidence that casts serious doubt on R. rubrum
RuBisCO promiscuity for the known RLP reactions.
1. Ashida H, Saito Y, Kojima C, Kobayashi K, Ogasawara N, Yokota A. (2003) A
functional link between RuBisCO-like protein of Bacillus and photosynthetic
RuBisCO. Science. 302, 286-90.
2. Imker HJ, Singh J, Warlick BP, Tabita FR, Gerlt JA. (2008) Mechanistic diversity in
the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCOlike protein from Rhodospirillum rubrum. Biochemistry. 47, 11171-3.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P50
Thymidylate Synthase Catalyzed H-Transfers: Role of Tunneling and
Dynamics in Different Enzyme catalyzed C-H activations
Zhen Wang, and Amnon Kohen
Department of Chemistry, University of Iowa, Iowa City, IA 52242
Examination of the nature of different bond activations along the same catalytic path is of
general interest in chemistry and biology. We compare the physical nature of two sequential
H-transfers in the same enzymatic reaction.1 Thymidylate synthase catalyzes a complex
reaction that involves many chemical transformations including two different C-H bond
cleavages, a rate-limiting C-H→C hydride transfer and a non-rate-limiting C-H→O proton
transfer (Figure 1).2 Although the large kinetic complexity imposes difficulties in studying the
proton transfer step, we are able to experimentally extract the intrinsic kinetic isotope effects
on both steps. In contrast with the hydride transfer, the intrinsic KIEs of the proton transfer
are temperature dependent. The results are interpreted within the framework of the Marcuslike model (also referred to as “environmentally coupled tunneling” or “full tunneling
model”). This interpretation suggests that this enzyme optimizes the donor-acceptor
geometries and dynamics for the slower and overall rate-limiting hydride transfer, which is
catalytically challenging. For the faster proton transfer, for which the catalytic effect (relative
to the solution reaction) is small, the geometries and dynamics are not optimized by the
enzyme. Similar physical examination of mutants revealed the role of active site residues in
different enzyme catalyzed C-H bonds activation.3,4
Figure 1. The proton and hydride transfers catalyzed by thymidylate synthase.
Reference
(1) Wang, Z., Kohen, A. J. Am. Chem. Soc. 2010, 132, 9820.
(2) Finer-Moore, J. S., Santi, D. V., Stroud, R. M. Biochemistry 2003, 42, 248.
(3) Hong, B., Maley, F., Kohen, A. Biochemistry 2007, 46, 14188.
(4) Hong, B., Haddad, M., Maley, F., Jensen, J. H., Kohen, A. J. Am. Chem. Soc. 2006,
128, 5636
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P51
Elucidating the Energetics of Active Site Loop Motion in
Formyl-CoA:Oxalate CoA Transferase
Sangbae Lee,¶ Mengen Chen,‡ Wei Yang‡ and Nigel G. J. Richards¶
¶
‡
Department of Chemistry, University of Florida, Gainesville, FL 32611, and
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306
This poster will describe recent work on computing the energetics of changing the
conformation of an active site loop1 in the “interlocked” homodimer of formyl-CoA:oxalate
CoA transferase.2 In addition to outlining the X-ray crystal structures of several intermediates
in the acyl transfer reaction catalyzed by FRC, which have delineated loop conformational
changes that mediate catalysis,3 the effect of site-specific mutations and substrate binding on
loop energetics and conformational preferences will be discussed.
(Left) Cartoon representation of the formyl-CoA transferase/CoA complex, showing the location of
the active site between the large and small sub-domains of monomers A and B, respectively. Protein
monomers are shown in green or blue ribbon representations. Bound coenzyme A is rendered in a
space-filling representation. Atom coloring: C, grey; N, blue; O, red; P, purple; S, yellow. (Right)
Cartoon showing superimposed active site tetrapeptide loops for the observed (2VJN) (cyan) and the
“open” (blue), “intermediate” (black) and “closed” (red) loop conformations calculated for the G260A
FRC mutant, together with the associated positions of the active site residues Trp-48, Tyr-59 and Asp169.
References
1. S. Lee, M. Chen, W. Yang and N. G. J. Richards (2010) J. Am. Chem. Soc. 132, 7252-7253.
2. S. Ricagno, S. Jonsson, N. G. J. Richards and Y. Lindqvist (2003) EMBO J. 22, 3210-3219.
3. C. Berthold, C. G. Toyota, N. G. J. Richards and Y. Lindqvist (2008) J. Biol. Chem. 282, 65196529.
Supported by the National Institutes of Health (DK61666 to N.G.J.R.)
and the National Science Foundation (MCB0919983 to W.Y.)
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P52
Discovery of a New Class of Quiescent Affinity Label:
4-Halopyridines Inactivate Dimethylarginine Dimethylaminohydrolase
Corey M. Johnson, Thomas W. Linsky, Joyce Er, Walter Fast*
Division of Medicinal Chemistry, College of Pharmacy;
University of Texas, Austin TX 78712; [email protected]
In an effort to develop novel covalent modifiers of dimethylarginine
dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of
“fragment”-sized inhibitors were identified using a high-throughput screen and tested for
time-dependent inhibition. One structural class, 4-halopyridines, are time- and concentrationdependent inactivators of DDAH. The inactivation mechanism of one example, 4-bromo-2methylpyridine (1), is characterized in detail. The neutral form is not very reactive with
excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent
and irreversible manner with the active-site Cys249 of the DDAH from Pseudomonas
aeruginosa. This active-site Cys is not particularly reactive (pKa ca. 8.8) and 1 does not
inactivate papain (Cys pKa ca. ≤ 4), suggesting that, unlike many thiol-targeted reagents, Cys
nucleophilicity is not a predominating factor in this inactivator’s selectivity. Rather, binding
and stabilization of the more reactive pyridinium form by a second moiety, Asp66, is required
for facile reaction. This constraint imparts a unique selectivity profile to the inactivator. To
our knowledge, 4-halopyridines have not previously been reported as protein modifiers, and
therefore represent a first-in-class example of a novel type of quiescent affinity label.
Cys249
Br
Br
pKa 4.75
N
1
S-
Br
Br
S
S
- Br -
+ DDAH
+
N
H
+
N
H
-O
+
N
H
N
H
O
-O
O
-O
O
Asp66
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P53
Mechanism of a disulfide-generating enzyme: the Quiescin-sulfhydryl
oxidase from Trypanosoma brucei
Vamsi K. Kodali, Benjamin A. Israel and Colin Thorpe
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716
Quiescin-sulfhydryl oxidase (QSOX) enzymes catalyze the net generation of disulfide bonds
in a wide range of non-fungal eukaryotes - from unicellular algae to humans. Their best
known substrates are unfolded reduced proteins:
Protein-(SH)2
+
O2
→
Protein-(S-S)
+
H2 O2
suggesting a role for these flavoproteins in oxidative protein folding. Here we report on a
QSOX from the human parasite Trypanosoma brucei. The domain structure and flow of
reducing equivalents for TbQSOX is as shown below:
Substrate protein (reaction 1) reduces the –CGAC- redox-active disulfide of the thioredoxin
(TRX) domain of TbQSOX. The protein substrate disengages, and then an inter-domain
movement (step 2) leads to reduction of the –CKEC- disulfide in the ERV/ALR domain.
Reduction of the flavin (step 3) by this proximal disulfide is followed by the reduction of
molecular oxygen to hydrogen peroxide (step 4). This poster explores facets of QSOX
structure and catalysis that involve the internal redox equilibrium steps 1-3. In particular, we
examine the consequences of changing the redox potential of the TRX CxxC motif for the
catalytic efficiency of the oxidase. Replacing the wild type sequence with CGPC makes the
thioredoxin domain of TbQSOX more reducing; conversely CPHC at the TRX domain
represents a more oxidizing site for input of reducing equivalents in step 1. Perturbation of
the balance between the three redox centers found in the wild-type protein has a major effect
on steady-state and pre-steady state kinetics and, surprisingly, alters the specificity of the
enzyme towards model thiol substrates. Evidence will be presented that the formation of an
inter-domain mixed disulfide bond (in step 2) is stabilized by a charge-transfer interaction
with the FAD (in step 3). The insights gained from this small trypanosomal QSOX will be
discussed in the context of the larger two-thioredoxin QSOXs from vertebrates.
This work was supported by NIH Grant GM26643.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
- but they are not found in fungi. Please prepare your abstract using this form.
P54
Two mechanisms for UDP-2,3-diacylglucosamine hydrolysis in lipid A
biosynthesis
Hayley E. Young1, Louis E. Metzger IV2, John K. Lee2, Robert M. Stroud2, Christian R. H.
Raetz1
1
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710
2
University of California San Francisco, San Francisco CA 94158
The outer-leaflet of the outer membrane of Gram-negative bacteria is composed of
lipopolysaccharide (LPS), a polysaccharide attached to the membrane via a hexa-acylated
saccharolipid. This anchoring molecule, called lipid A, is required for growth in nearly all
Gram-negative bacteria, thus making its biosynthesis an excellent target for new antibiotics
(1). Most of the nine enzymes in the lipid A biosynthetic pathway are conserved. However,
the fourth step of the pathway, involving the conversion of UDP-2,3-diacylglucosamine
(UDP-DAGn) to 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP can be carried out by
either LpxH (2) or LpxI (3). These proteins are unrelated enzymes and are never found in the
same organism. Moreover, they use different mechanisms to catalyze the formation of lipid
X, with LpxH catalyzing the attack of water on the α phosphate and LpxI on the β phosphate
of the pyrophosphate bond in UDP-DAGn (2, 3). LpxH is primarily found in γ- and εproteobacteria and bioinformatic analysis classifies it as a member of the metal-dependent
phosphoesterase (MDP) superfamily (Pfam00149) (2). We have cloned and over-expressed
the Haemophilus influenzae ortholog of LpxH (HiLpxH) in E. coli. HiLpxH has been
purified to 95% homogeneity using NiNTA and gel filtration chromatography. Like other
enzymes of the MDP family, HiLpxH appears to utilize divalent cations in catalysis, as Mn2+
is required for activity in vitro. Alanine point mutants of HiLpxH indicate the importance of
the residues in the MDP motif (DXHX~25GDXXDRX~25GNHD/E) for HiLpxH activity and
other residues specific to LpxH orthologs also seem to be important in enzyme function.
Mass spectrometry reveals that lipid X co-purifies with HiLpxH. Interestingly, this has also
been observed with C. crescentus LpxI, the crystal structure of which was recently determined
at 2.5 Å (L. Metzger, J. Lee, R. Stroud, and C. Raetz, in preparation), suggesting that while
the enzymes show no sequence similarity, they may nevertheless share a product-binding site.
Overall, this work sets the scene for further structural and mechanistic studies of LpxH and
LpxI. This work is supported by NIH grant GM051310 to C.R.H.R and Specialized Center
grant of the Protein Structure Initiative U54 GM074929-01 to R.M.S.
1.
2.
3.
Raetz, C. R. H., Reynolds, C. M., Trent, M. S., and Bishop, R. E. (2007) Lipid A
modification systems in gram-negative bacteria, Annu Rev Biochem 76, 295-329.
Babinski, K. J., Ribeiro, A. A., and Raetz, C. R. H. (2002) The Escherichia coli gene
encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis, J
Biol Chem 277, 25937-25946.
Metzger, L. E. t., and Raetz, C. R. H. An alternative route for UDP-diacylglucosamine
hydrolysis in bacterial lipid A biosynthesis, Biochemistry 49, 6715-6726.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P55
Enzymatic Study and Chemical Inhibitor of the Histone Acetyltransferase
Tip60
Jiang Wu, Nan Xie, Emilia N. Elangwe, Chao Yang and Yujun George Zheng*
Department of Chemistry, Molecular Basis of Disease Program
Georgia State University, 30303, [email protected]
Tip60 (HIV-1 TAT-interactive protein, 60 kDa) is a key member of the MYST family of
histone acetyltransferases (HATs) and plays important functions in many cellular processes
and human diseases. We report here both substrate-based analog inhibitors and small
molecule inhibitors for Tip60. In the first strategy, we designed, synthesized and evaluated a
series of substrate-based analogs for the inhibition of Tip60. The structures of these analogs
feature that coenzyme A is covalently linked to the side chain amino group of the acetyl
lysine residues in the histone peptide substrates. These bisubstrate analogs exhibit stronger
potency in the inhibition of Tip60 compared to the small molecules curcumin and anacardic
acid. The substrate-based analog inhibitors will be useful mechanistic tools for analyzing
biochemical mechanisms of Tip60, defining its functional roles in particular biological
pathways, and facilitating protein crystallization and structural determination.
We also performed a virtual screening using the crystal structure of Esa1 (the yeast
homolog of Tip60) and 105 small molecules were selected as potential hits. Then, the
radioactive HAT assay was used for experimental screening. From the first-round screening,
we focus on a series of small molecules that bear structure similarity with JWu-10 to identity
lead inhibitors. We also tested the selectivity of this new class of Tip60 inhibitors. IC50 was
compared to find out the structure-activity relationship. The discovery of new small molecule
inhibitors provides important chemical tools for functional study of Tip60 and is of great
potential to be developed into valuable anticancer agents.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P56
Engineering E. coli for new chemical function
Brooks B. Bond-Watts and Michelle C.Y. Chang
Departments of Chemistry and Molecular & Cell Biology, University of California, Berkeley
94720-1460
Synthetic biology approaches allow us to mix and enzymes from different organisms in order
to create new synthetic pathways inside living organisms for the scalable production of target
small molecules. Although methods for DNA sequencing and assembly are rapidly
expanding, our ability to construct de novo pathways remains limited by insufficient
understanding of how chemistry works inside a living cell. Our group is interested in using
synthetic biology as a platform to understand the molecular principles needed to reprogram
the cell for new biosynthetic function. We have focused on the construction of pathways for
the microbial synthesis of second-generation biofuels, as their sustainable production relies on
developing new fuel structures with improved molecular properties compared to ethanol with
near theoretical yields from glucose that can compete with mature yeast fermentation
processes. Towards these goals, we have built a robust pathway for the production of nbutanol from individual enzyme components and explore how enzyme mechanism can be
used as a kinetic control element to push a reversible pathway to high yielding production of
second-generation biofuels (> 4 g/L). Furthermore, experiments indicate that our engineered
pathway operates within the dynamic range that allow fuel titers to report on cellular
decisions in carbon fate and can possibly used as a synthetic phenotype for the discovery of
new metabolic and regulatory elements that control central metabolism.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P57
Characterization of enzymes involved in the degradation of
2-aminoethylphosphonate in Sinorhizobium meliloti:
phosphonoacetaldehyde dehydrogenase PhnY and phosphonoacetate
hydrolase PhnA
Svetlana A. Borisova,1,5 Vinayak Agarwal,1,2 Nurul Zulkepli,3 Satish K. Nair,1,2,3,*
Wilfred A. van der Donk,1,3,5,* and William W. Metcalf1,4,*
1
Institute for Genomic Biology, 2Center for Biophysics and Computational Biology,
3
Department of Chemistry, 4Department of Microbiology, 5Howard Hughes Medical Institute
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA;
Phosphonates, carbon-phosphorus (C-P) bond-containing organic molecules, can be utilized
by a variety of microorganisms as a source of phosphorus, carbon, and nitrogen via several
pathways. The broad specificity C-P lyase enzyme allows catabolism of a wide variety of
structurally-diverse phosphonates and is widespread among bacteria. In addition, there are at
least three other phosphonate-specific catabolic pathways that do not involve C-P lyase. In
Sinorhizobium meliloti 1021 a number of phosphonates are catabolized via the C-P lyase
pathway, but an alternative C-P lyase-independent pathway for degradation of 2aminoethylphosphonate (2-AEPn) in S. meliloti has been proposed (1). We identified a gene
cluster, presumably responsible for the breakdown of 2-AEPn to phosphate and acetate, that
contains genes encoding for a putative 2-AEPn:pyruvate aminotransferase (Smb21537), an
aldehyde dehydrogenase (phnY), and a phosphonoacetate hydrolase (phnA). Biochemical
characterization of recombinant PhnY and PhnA proteins confirmed their proposed catalytic
functions. PhnY is an NADP-dependent dehydrogenase converting phosphonoacetaldehyde
(originating from transamination of 2-AEPn) to phosphonoacetic acid. Phosphonoacetate is
then converted to acetate and inorganic phosphate by the metal-dependent phosphonoacetate
hydrolase PhnA. Crystal structures of PhnA suggest a catalytic mechanism similar to that of
enzymes belonging to the alkaline phosphatase superfamily. The kinetic properties of PhnA in
the presence of different divalent metal ions were also investigated. This study reports the
characterization of a novel pathway for 2-AEPn degradation, and provides greater insight into
the microbial metabolism of phosphonates.
References
1. Parker, GF; Higgins, TP; Hawkes, T; Robson, RL; J. Bacteriol. 1999, 181, 389.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P58
Suppression of a polymerase general acid mutant:
Consequences on replication speed and polymerase fidelity
Jamie J. Arnold, Madhumita Yennawar, Yan Zhao, Eric D. Smidansky and Craig E. Cameron
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, PA 16802
Catalysis by all four classes of nucleic acid polymerases is enhanced by use of a general acid.
In most polymerases, the general acid is a lysine. In multi-subunit RNA polymerases, the
putative general acid is a histidine. We hypothesized that the lower pKa of histidine relative
to lysine permits pyrophosphorolysis to be used more efficiently for error correction and/or
release of stalled transcription complexes. To test this, we engineered a poliovirus
polymerase that had its general acid (Lys-359) converted to histidine. The activity of this
polymerase was reduced 10-fold in vitro and in vivo; however, the fidelity was indeed higher
than observed for wild-type. We next used viral genetic selection to isolate a poliovirus
mutant expressing a polymerase variant that retained histidine but exhibited wild-type,
polymerase activity. This approach identified a polymerase that changed Ile-331 to Phe. We
find that this polymerase is as active as wild-type polymerase. Additional experiments of the
more active His-359 derivative suggest that the nature of the general acid influences
replication speed and polymerase fidelity. Our hope is that we have now created the first
panel of polymerases that change fidelity without decreasing activity, thus permitting a true
assessment of the role of fidelity on viral replication, evolution, population dynamics and
pathogenesis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P59
Identification of PODANSg2158 from P. anserina as a Nitroalkane Oxidase
and Comparison with the Enzyme from F. oxysporum
José R. Tormos and Paul F. Fitzpatrick
Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX,
78229
The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the
oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones.
NAO is homologous to acyl-CoA dehydrogenase (ACAD), so that several fungal genes
annotated as coding for hypothetical proteins or putative ACADs are likely to instead be for
NAOs. Using the active site triad in NAO, Ser276, Asp402 and Arg409, as a probe, the gene
PODANSg2158 from Podospora anserina was identified as a potential NAO. We have been
able to express and purify the recombinant protein. The enzyme is a flavoprotein that
catalyzes the oxidation of nitroalkanes, although the substrate specificity is slightly different
from that of the F. oxysporum enzyme. The kcat/Km-pH profile with nitroethane shows a pKa
of 5.9 compared to the value of 6.9 for the F. oxysporum enzyme; this pKa is assigned to
Asp399, the active site base. Mutation of Asp399 to asparagine decreases the kcat/Km value for
nitroethane over two orders of magnitude. The R406K and S373A mutations decrease this
kinetic parameter by 64 and 3-fold, respectively. The structure of PODANSg2158 has been
determined at a resolution of 2.0 Å, confirming its identification as an NAO.
Crystal structures of mutant enzymes with nitroalkanes bound show that the oxygen of
the nitro group interacts with the 2’-hydroxyl group of the FAD1. To determine the
importance of this interaction, the enzymes from both organisms were reconstituted with 2’deoxy-FAD. In both cases, no activity could be detected using nitroethane as the substrate.
(1)
Heroux, A.; Bozinovski, D. M.; Valley, M. P.; Fitzpatrick, P. F.; Orville, A. M.
Biochemistry 2009, 48, 3407.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P60
Inhibition by Nitric Oxide of Catalysis by Oxalate Decarboxylase
MARIO EDGAR G. MORAL†, Chingkuang K. Tu‡, Witcha Imaram†,
Alexander Angerhofer†, David N. Silverman‡, and Nigel G. J. Richards†
†
Department of Chemistry, University of Florida, Box 117200, Gainesville, FL 32611.
‡
Departments of Pharmacology & Biochemistry, College of Medicine, University of Florida,
Box 100267 HSC 1600 SW Archer Rd, Gainesville, FL 32610.
Oxalate decarboxylase (OxDC) is a manganese-containing enzyme, which catalyzes
the non-oxidative breakdown of monoprotonated oxalate to carbon dioxide and formate. The
reaction requires dioxygen, even though it remains unclear as to its role in the overall
mechanism, and where it binds in the enzyme. Studying the effects of an oxygen analogue
such as nitric oxide (NO) on the catalytic reaction, may shed light on the location and role of
the dioxygen in the enzyme during catalysis. However, traditional endpoint assays used to
measure the kinetics of this enzyme preclude the ability to look into the progress of the
reaction as the products are formed. Here we report the use of membrane inlet mass
spectrometry as a continuous and real-time method of monitoring catalysis by OxDC from B.
subtilis, by observing the accumulation of CO2 in solution from its mass peak m/z 44 (or m/z
45 for 13CO2). NO was generated in solution from NONOates, and complete inhibition by
micromolar NO (Ki = 40 ± 10 µM) was observed. The inhibition was reversed after a time lag
of several minutes by the addition of O2. Electron paramagnetic resonance of a rapidly frozen
reaction mixture showed that the multiplet splittings of Mn(II) on X-band were not perturbed
by the presence of NO. This suggests that although NO is an inhibitor, it does not directly
bind to the Mn(II) site with the smallest fine structure (|D| = 1200 MHz) in the enzyme.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P61
Studies on the kinetic sequence of the Mycobacterium
tuberculosis DHFR
Clarissa M. Czekster, An Vandemeulebroucke, John S. Blanchard
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461, United States.
Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate,
generating tetrahydrofolate, a key molecule for one-carbon metabolism, and essential for
DNA synthesis1. Although the M. tuberculosis DHFR (MtDHFR) is considered a promising
target for anti-tubercular drugs, little information is available about the catalytic cycle of this
reaction. In this work, the kinetic sequence of the MtDHFR was determined, by conducting a
complete pre steady-state characterization of the MtDHFR-catalyzed reaction. Single turnover
experiments revealed that the chemical step is occurring at 6 s-1, at least three orders of
magnitude smaller than observed for DHFRs from other organisms2. Additionally, the
presence of a burst of product formation indicated that the kcat-determining step takes place
after the chemical step. Stopped-flow fluorescence combining binding and competition
experiments was employed to measure kinetic rate constants for binding and dissociation of
the two substrates and the two products. These experiments revealed that, after the chemical
step, the MtDHFR follows two parallel pathways for product release. Moreover, the
temperature dependence of the chemical step was studied, and an activation energy of 13.6
kcal mol-1 for the chemical step was determined. Based on the kinetic data obtained, an
energy profile for the catalytic cycle was generated, illustrating the random product release.
Future studies include the evaluation of the rate of chemistry at different pHs, and the
characterization of mutants to better understand the slow rate of the chemical step.
References
1. Kompis, I.M., Islam, K., Then, R.L. (2005) DNA and RNA Synthesis: Antifolates. Chem.
Rev. 105, 593-620.
2. Fierke, C.A., Johnson,K.A., Benkovic, S.J. (1987) Construction and Evaluation of the
Kinetic Scheme Associated with Dihydrofolate Reductase from Escherichia coli.
Biochemistry 26, 4085-4092.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P62
Design, Synthesis and Evaluation of Quinolone-based Inhibitors of
E. coli PBP5
A. G. Shilabin,a B. Jayaram,b and R. F. Pratta*
a
b
Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA
Department of Chemistry, Indian Institute of Technology Delhi , Hauz Khas, New Delhi,
110016, India
Penicillin-binding proteins (PBPs) are important bacterial enzymes that carry out the final
steps of bacterial cell wall assembly. DD-Transpeptidases accomplish the essential peptide
cross-linking step of the cell wall. To date, most attempts to discover effective inhibitors of
PBPs, apart from β-lactams, have failed. Therefore, the need for new classes of efficient
inhibitors remains. In E. coli, the low molecular mass class A PBP5 is the most abundant and
well-characterized DD-peptidase. Beginning with fragment-based docking studies, we have
designed and synthesized a series of 4-quinolone analogs as potential inhibitors of PBPs. We
describe the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the
major classes, highlighting their inhibitory activity against PBP5 from E. coli.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P63
Inhibition of Bacterial Phosphopantothenoylcysteine Synthetase
James D. Patrone, Jiangwei Yao, Jennifer L. Meagher, Jeanne A. Stuckey, and
Garry D. Dotson.
University of Michigan, College of Pharmacy, Department of Medicinal Chemistry,
and the Life Sciences Institute, Ann Arbor, MI 48109
Phosphopantothenoylcysteine (PPC) synthetase (CoaB; EC 6.3.2.5) catalyzes the
condensation of phosphopantothenate (PPA) and L-cysteine in coenzyme A (CoA)
biosynthesis, and exist in prokaryotes in both a bifunctional and monofunctional form. The
former is expressed as a protein fusion with PPC decarboxylase (CoaC; EC 4.1.1.36), which
catalyzes the subsequent biosynthetic step in the synthesis of CoA. Whether monofunctional
or bifunctional, CoaB catalysis in bacteria is exclusively cytidine triphosphate-dependent. We
have synthesized a series of inhibitors based upon the phosphopantothenoyl cytidylate,
mixed-anhydride formed during bacterial CoaB catalysis.1 These compounds display potent
inhibition towards both monofunctional and bifunctional CoaB, and up to 1000 fold
selectivity of inhibition over human CoaB. The most potent inhibitor shows a slow onset,
tight binding mode of inhibition with a Ki of 24 nM.2
To determine the structural basis for the variance in potencies, the above
inhibitors have been co-crystallized with the native CoaB domain of E. coli and their x-ray
structures determined. The nucleotide binding pocket shows very little difference among the
three inhibitor-bound structures and is analogous to the published intermediate-bound E. coli
N210D PPCS structure.1 The decreased inhibitor potency seen with the sulfamate and
cyclophosphate containing inhibitors, compared to the phosphodiester mimic, can be
determined structurally as arising from the lack of salt bridge formation between the
sulfamate isostere and Lys289, and significant steric clash of the gem dimethyl of the
cyclophosphate moiety that keeps the Asp354 to Ala368 binding flap from closing down on
the PPA binding pocket.
1.
Stanitzek, S.; Augustin, M. A.; Huber, R.; Kupke, T.; Steinbacher, S., Structural basis
of CTP-dependent peptide bond formation in coenzyme A biosynthesis catalyzed by
Escherichia coli PPC synthetase. Structure 2004, 12, (11), 1977-1988.
2.
Patrone, J. D.; Yao, J.; Scott, N. E.; Dotson, G. D., Selective inhibitors of bacterial
phosphopantothenoylcysteine synthetase. J Am Chem Soc 2009, 131, (45), 16340-16341.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P64
Determination of the crystal structure of a two-domain NRPS fusion
protein, EntEB, from the enterobactin synthetic cluster of Escherichia coli.
Jesse A. Sundlov1, Eric J. Drake1, Courtney C. Aldrich2, and Andrew M. Gulick1.
1
Hauptman-Woodward Medical Research Institute, State University of New York at Buffalo,
Department of Structural Biology, 700 Ellicott St., Buffalo, NY 14203, 2Center for Drug
Design, University of Minnesota, Academic Health Center, Minneapolis, MN 55455.
The Escherichia coli enterobactin siderophore synthetic cluster includes a two-module nonribosomal peptide synthetase (NRPS), composed of EntB, EntE, and EntF, that catalyzes the
formation of enterobactin from three serine molecules and three molecules of 2,3dihydroxybenzoic acid (DHB). EntE, acting as a free-standing NRPS adenylation domain,
combines DHB and ATP to form an aryl adenylate and inorganic pyrophosphate. In a second
reaction, EntE transfers the DHB molecule to the pantetheine cofactor of the EntB carrier
protein domain. We have created a chimera of the carrier protein domain of EntB and the full
length EntE enzyme. Implementing a substrate mimic for the EntE adenylation domain, we
have successfully crystallized this fusion protein. Here we present the crystal structure of this
two-domain NRPS complex. The structure gives insight into the interdomain architecture and
also supports our previous hypothesis that domain rearrangements within the adenylation
domain are mechanistically important to the NRPS assembly line.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P65
Probing the Mechanism of Phenylalanine Hydroxylase
Using Rapid-Mixing Techniques with High-Resolution Mass Spectrometry
Kenneth M Roberts and Paul F Fitzpatrick
Department of Biochemistry
University of Texas Health Science Center at San Antonio
7703 Floyd Curl Dr.
San Antonio, TX 78229-3900
Phenylalanine hydroxylase (PheH), a member of the aromatic amino acid hydroxylase family,
catalyzes the oxidation of phenylalanine to tyrosine, the precursor to dihydroxyphenylalanine
(DOPA) and the catecholamine neurotransmitters. Defects in PheH form the basis of the
metabolic diseases hyperphenylalaninemia (HPA) and phenylketonuria (PKU), characterized
by mental retardation, impaired cognitive development and seizures.1,2 PheH is a non-heme
iron monooxygenase whose catalysis is dependent on the presence of the reducing substrate,
tetrahydrobiopterin. The proposed mechanism involves the formation of an iron-peroxypterin
which decays to a 4a-hydroxypterin and an Fe(IV)O responsible for amino acid
hydroxylation.3 Aside from steady-state rates of turnover, rate constants of the reaction are not
known. Further, dehydration of the 4a-hydroxypterin to a dihydropterin is nonenzymatic and
rapid,4 making detection difficult, and there is no experimental evidence for a peroxypterin
intermediate either in the enzymatic reaction or in the autooxidation reaction of
tetrahydropterins. In this study, we employed rapid-reaction techniques to monitor the
reaction under presteady-state conditions. Rapid-mixing chemical-quench experiments were
performed monitoring tyrosine formation. Reactions were performed by mixing a solution of
PheHΔ117, the catalytic domain of PheH, and phenylalanine in buffer with a solution of
6-methyltetrahydropterin (6MPH4). At 5 °C, a burst-phase is observed within the first second
(kburst and klinear of 7.0 and 1.2 s-1, respectively), indicating a rate-determining product release.
Additionally, a Thermo Scientific OrbiTrap high-resolution mass spectrometer with a custom
nano-spray source incorporating a continuous-flow mixer with Peltier control was employed
monitoring the reaction under similar conditions at times of approx. 0.5 and 2.5 s. The high
resolution of the mass analysis allows us to distinguish tyrosine (M+1 exact mass 182.0812)
and 6MPH4 (M+1 exact mass 182.1036). Further, we observe the reaction product,
4a-hydroxy-6-methyltetrahydropterin (M+1 exact mass 198.0986), a clear example of the
ability of the assay to monitor early reaction times and short-lived intermediates. Though a
peroxypterin intermediate was not observed in the enzymatic reaction, autooxidation of
6-methyltetrahydropterin produces a reaction product at m/z 214.0933, which may be
attributed to 6-methyl-4a-hydroperoxytetrahydropterin (M+1 exact mass 214.0935).
References
(1)
Williams, R. A.; Mamotte, C. D. S.; Burnett, J. R. Clin Biochem Rev 2008, 29, 31.
(2)
Scriver, C. R.; Waters, P. J. Trends Genet. 1999, 15, 267.
(3)
Fitzpatrick, P. F. Biochemistry 2003, 42, 14083.
(4)
Bailey, S. W.; Rebrin, I.; Boerth, S. R.; Ayling, J. E. JACS 1995, 117, 10203.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P66
Identification of the Enzyme That Catalyzes the Final Step in the
Biosynthesis of Queuosine
Zachary D. Miles, Reid M. McCarty, Gabriella Molnar & Vahe Bandarian
University of Arizona, Department of Chemistry and Biochemistry, 1041 E. Lowell St.,
Tucson, AZ 85721-0088
Queuosine (Q) is a hypermodified base which is present at the wobble position in the 3′GUN-5′ anticodon loop of Asn, Asp, His, and Tyr tRNA. It is synthesized de novo in
prokaryotes, in contrast to eukaryotes, where it is digested as the free-base queuine and
exchanged for guanine in a one step transglycosolation reaction catalyzed by the enzyme Tgt.
While the modification was discovered nearly 40 years ago (1), the role queuosine plays
within biological systems is still unclear due to the lack of distinct observable phenotypes.
Yet, because the modification is evolutionarily conserved throughout all domains of life (2),
the biological role is of great importance. Queuosine contains the 7-deazapurine core structure
observed in secondary metabolites in the order actinomyces. The structure is derived from a
purine precursor where the nitrogen in position seven of the ring is replaced by a carbon.
Previous work in the Bandarian lab has led to elucidation of the first four steps in the
biosynthetic pathway to the small molecule preQ0 (3), which is a common intermediate in the
biosynthesis of multiple 7-deazapurine secondary metabolites. While the next three steps in
the pathway of queuosine had been deduced previously, the final step had yet to be
determined. To search for this enzyme, we utilized the Keio Collection, which is a library of
isogenic variants of E. coli K12, where all of the ~ 4000 nonessential genes had individually
been replaced by a kanamycin resistance cassette. Each mutant with a deletion of a gene of
unknown function (~1800) was cultured, whole cell RNA was purified, digested to the
nucleoside level and analyzed using LC-MS. This led to identification of a mutant that
accumulates epoxyqueuosine (oQ) and contains no queuosine. This poster will summarize
current progress on the in vitro reconstitution of the oQ reductase activity.
References
1. S. Nishimura, Structure, Biosynthesis, and Function of Queuosine in Transfer RNA. Prog.
In Nuc. Acid Res. and Mol. Biol. 28, 49-73 (1983).
2. J. R. Katze, B. Basile, J. A. McCloskey, Queuine, a modified base incorporated
posttranscriptionally into eukaryotic transfer RNA: wide distribution in nature. Science 216,
55-56 (1982).
3. R. M. McCarty et al. The deazapurine biosynthetic pathway revealed: in vitro enzymatic
synthesis PreQ0 from guanosine 5'-triphosphate in four steps. Biochemistry 48, 38473852 (2009).
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P67
The chemistry of DXP synthase: Implications for selective inhibitor design
Francine Morris and Caren L. Freel Meyers
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine,
Baltimore, MD, 21205.
Many human pathogens rely upon the methylerythritol phosphate (MEP) pathway for
biogenesis of the essential isoprenoid bioprecursors isopentenyl pyrophosphate (IPP) and
dimethylallyl pyrophosphate (DMAPP). Our long term goal is to understand catalysis of these
intriguing biosynthetic enzymes toward the development of new anti-infective agents. 1Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the MEP pathway to
form DXP from pyruvate and glyceraldehyde 3-phosphate (GAP). Its unique structure and
mechanism distinguish DXP synthase from its homologs, making it a particularly interesting
target. We have hypothesized that DXP synthase exhibits distinctive catalytic activities,
offering opportunities to discover new reactions and probe unique features of catalysis toward
guiding selective inhibitor design. Here, we present substrate specificity studies that reveal a
new function of DXP synthase as a catalyst in C-N bond formation. Our results further
highlight a remarkable affinity of the enzyme for naphthol-based substrates. Current efforts
are focused on delineating this scaffold as a starting point for selective inhibitor design toward
targeting non-mammalian isoprenoid biosynthesis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P68
The Parts Aren’t Interchangeable: Installing a Proton-Transfer Network
into a Class 1A Dihydroorotate Dehydrogenase
Claudia A. McDonald and Bruce A. Palfey
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI
Dihydroorotate dehydrogenases (DHODs) are flavin-containing enzymes that catalyze the
conversion of dihydroorotate to orotate. There are three classes of DHODs: Class 1A, Class
1B, and Class 2. Class 1 has a cysteine as a base, while Class 2 has a serine. The Class 1A
enzyme from Lactococcus lactis Cys133Ser mutant is ~105-fold slower than wild-type (1).
Class 1A enzymes do not have a proton-transfer network that connects the active site base to
bulk solvent but Class 2 enzymes do (2). Can installing a proton-transfer network restore
rapid reduction to the Cys133Ser Class 1A mutant enzyme?
The Class 2 proton-transfer network is conserved in Class 2 enzymes. The Class 2 protontransfer network is made up of Ser175, Thr178, and Thr178 is in contact with Phe115. We
have shown the proton-transfer network in the Class 2 enzyme from Escherichia coli
contributes to speeding the reduction of FMN (2). Class 1A enzymes have conserved Cys,
Val, and Leu in the corresponding positions of the Class 2 proton-transfer network. Our
results show that adding a proton-transfer network to Class 1A did not restore rapid reduction
but drastically worsened the rate of reduction. Interestingly a key residue, Leu71, was found
to be critical for the binding of 3,4-dihydroxybenzoate, a ligand specific for Class 1A
enzymes, but not 3,5-dihydroxybenzoate, the other ligand that is specific for Class 1A
enzymes.
References
1. Fagan, R.L., Jensen, K.F., Bjornberg, O., and Palfey, B.A. (2007) Mechanism of
flavin reduction in the Class 1A dihydroorotate dehydrogenase from Lactococcus
lactis. Biochemistry 46, 4028-4036.
2. Kow, R.L., Whicher, J.R., McDonald, C.A., Palfey, B.A., and Fagan, R.L. (2009)
Disruption of the proton relay network in the Class 2 dihydoroorotate dehydrogenase
from Escherichia coli. Biochemistry 48, 9801-9809.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P69
Characterization of the Putative Diels-Alderase LovB CON
Joel J Bruegger1, Brian Ames1, Suzanne Ma4, Jesse Li5, John Vederas5, Yi Tang,4 Sheryl
Tsai1,2,3
1
Department of Molecular Biology and Biochemistry, 2Department of Chemistry,
3
Department of Pharmaceutical Sciences, University of California, Irvine, California, 92697,
USA. 4Department of Chemistry and Biomolecular Engineering, University of California, Los
Angeles, California, 90095, USA. 5Department of Chemistry, University of Alberta,
Edmonton, Alberta, T6G 2G2, Canada.
Lovastatin is one of the best selling cholesterol-lowering drugs in the world, and it has
been used as the chemical core for developing additional highly effective anti-cholesterol
drugs1,2,3. The lovastatin nonaketide synthase (LovB) from Aspergillus terreus is responsible
for initiating the biosynthesis of lovastatin4,5, which has been proposed to exhibit a novel
Diels-Alderase activity during the biosynthesis6,7,8. The condensation domain (CON) of the
PKS LovB is proposed to be responsible for the Diels-Alderase activity based on studies
where the deletion of LovB CON abolishes the formation of LovB’s natural product,
dihydromonacolin L. Instead, the CON-deleted mutant produced a hexaketide, which is the
proposed immediate precursor to the Diels-Alder reaction9. The lack of comprehensive data
has prevented a confirmation about the role of LovB CON domain. If proven true, the CON
domain would be the first identified Diels-Alderase involved in polyketide biosynthesis.
Studies that combine chemical synthesis, enzymatic assays and structural characterization
are necessary to understand any potential Diels-Alderase activity for future natural product
studies.
References
1. Krukemyer JJ & Talbert RL (1987) Lovastatin: a new cholesterol-lowering agent. in
Pharmacotherapy, pp 198-210.
2. Manzoni M & Rollini M (2002) Biosynthesis and biotechnological production of statins
by filamentous fungi and application of these cholesterol-lowering drugs. in Applied
microbiology and biotechnology, pp 555-564.
3. Tobert JA (2003) Lovastatin and beyond: the history of the HMG-CoA reductase
inhibitors. in Nature reviews Drug discovery, pp 517-526.
4. Hendrickson L, et al. (1999) Lovastatin biosynthesis in Aspergillus terreus:
Characterization of blocked mutants, enzyme …. in Chemistry & Biology, pp 429-439.
5. Kennedy J, et al. (1999) Modulation of polyketide synthase activity by accessory proteins
during lovastatin biosynthesis. in Science, pp 1368-1372.
6. Hutchinson CR, et al. (2000) Aspects of the biosynthesis of non-aromatic fungal
polyketides by iterative polyketide synthases. in Antonie Van Leeuwenhoek, pp 287-295.
7. Ma SM & Tang Y (2007) Biochemical characterization of the minimal polyketide
synthase domains in the lovastatin nonaketide synthase LovB. in FEBS J, pp 2854-2864.
8. Witter DJ & Vederas JC (1996) Diels-Alder catalyzed cyclization during biosynthesis of
lovastatin. in J Org Chem, pp 2613-2623.
9. Ma SM, et al. (2009) Complete reconstitution of a highly reducing iterative polyketide
synthase. in Science, pp 589-592.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P70
Structural Analysis of the Isochorismate-Pyruvate Lyase
from Pseudomonas aeruginosa
Andrew Ouellette, Jose Olucha, Qianyi Luo, Roberto De Guzman and Audrey L. Lamb
Molecular Biosciences, University of Kansas, Lawrence, KS, 66045
Understanding the origin of catalytic power is an important question in the field of
enzymology today.1-2 The isochorismate-pyruvate lyase of Pseudomonas aeruginosa, PchB,
serves as a model to answer fundamental questions. The physiological activity of PchB, a
structural homologue of E. coli chorismate mutase, is that of an isochorismate-pyruvate
lyase, but PchB also has adventitious chorismate mutase activity.3 Previous structural
analyses of PchB have shown a large conformational change in the active site loop upon
ligand binding, suggesting a potentially significant role for loop dynamics in catalysis.4
Substitution mutations at a single residue (K42) within the active site loop are sufficient to
modulate PchB activity by several orders of magnitude.5 In this study, we present the crystal
structures for wild-type PchB (1.46 Å resolution) and two mutants structures K42H (2.31 Å)
and K42E (1.84 Å) with the products of the lyase reaction (salicylate and pyruvate) bound in
the active site. Analysis of the crystal structures shows a conserved active site architecture
among the wild-type and PchB mutants. This strongly suggests that the differences in the
catalytic activities are due to the change in chemical nature of the residue at the 42 site. We
also present our preliminary work to analyze PchB loop dynamics by Nuclear Magnetic
Resonance (NMR) relaxation dispersion experiments.
References:
1. R. L. Schowen. PNAS (2003) 100, 11931-2.
2. Reviews: S. Borman. C&E News (2004) 88, 35-39. E. Wilson. C&E News (2000)78, 42-45.
3. C. Gaille, P. Kast, D. Haas. J Biol Chem (2002) 277, 21768-75.
4. J. Zaitseva, Q. Luo, K.L. Olechoski, A.L. Lamb. (2006) J Biol Chem 281, 33441-9.
5. Q. Luo, J. Olucha, A. L. Lamb. Biochemistry (2009) 48, 5239-5245.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P71
A Site Under Selective Pressure in the LexA Repressor is Required for
Control of the DNA Damage Response
Marciano DC, Katsonis P, Adikesavan AK, Herman C, and Lichtarge O
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
The LexA repressor of the bacterial DNA damage response or its interaction with the RecA
recombinase may be good drug targets for combating the emergence of antibiotic resistance. LexA
structure-function relationships can help guide the development of LexA inhibitors. LexA has several
crystal structures and functional studies available and yet, the mechanism of RecA-mediated LexA
hydrolysis is uncertain. Here we use a computational tool for sequence analysis to identify a new
functional site on LexA and provide genetic evidence indicating its importance in repressor function.
LexA residues were ranked according to each position’s variation pattern as it relates to the protein
family tree using the Evolutionary Trace program (http://mammoth.bcm.tmc.edu/). Core residues of
the known auto-proteolytic active site, cleavage site and DNA binding site are within the top 10th
percentile; indicating accuracy in the identification of functional sites (see figure). Eighteen highly
ranked residues were targeted for site-directed mutagenesis and their effect upon resistance to DNA
damage and in vivo repressor function was measured. Five of six mutations in a site adjacent to the
dimerization interface improved resistance to DNA damage; suggesting a constitutive or sensitized
response to DNA damage. This site contributes towards repressor function and steady-state protein
expression levels as indicated by a LexA-repressed GFP reporter assay and Western blot analysis. This
modulation site on LexA may influence RecA-mediated hydrolysis and prove amenable to
pharmacological inhibition that can delay the appearance and spread of antibiotic resistance
determinates.
Supported by training fellowships from the National Institutes of Health and the National Library of
Medicine to the Keck Center Pharmacoinformatics and Interdisciplinary Bioscience Training
Programs of the Gulf Coast Consortia to PK and DCM (NIH Grant T90DK070109-05 & NLM Grant
T15LM007093).
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P72
Characterization of Ceriporiopsis subvermispora Bicupin Oxalate Oxidase
Expressed In Pichia pastoris
Kelsey Ubertoa, Eric Hoffer a, Patricia Moussatcheb, Alexander Angerhoferb, Witcha Imaramb,
Nigel G. J. Richardsb, Ellen W. Moomawa
a
Department of Chemistry and Biochemistry, Kennesaw State University, 1000 Chastain Road,
Kennesaw, GA 30144-5588.
b
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 326117200.
Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon
dioxide in a reaction that is coupled with the formation of hydrogen peroxide (Scheme below).
Although there is currently no structural information available for oxalate oxidase from
Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it
is the first manganese-containing bicupin enzyme identified that catalyzes this reaction.
Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate
decarboxylases (OxDC).
CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to
support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with
the coordination environment expected from homology modeling with known X-ray crystal
structures of oxalate decarboxylase from Bacillus subtilis. EPR spin-trapping experiments
support the existence of an oxalate-derived radical species formed during turnover. We have
determined that acetate and a number of other small molecule carboxylic acids are competitive
inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction
suggests that the dominant contribution to catalysis comes from the monoprotonated form of
oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be
unprotonated.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P73
Chemical Model for the Action of a Single Catalytic Residue, Arginine, at
Active Site of Uroporphyrinogen Decarboxylase
Charles A. Lewis, Jr. and Richard Wolfenden
Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill
Uroporphyrinogen Decarboxylase (UroD, EC 4.1.1.37) catalyzes the first committed step in
the biosynthesis of heme, chlorophyll, and cytochromes, in which an acetate group on each of
the 4 unconjugated pyrrole rings is decarboxylated to a methyl group in a single enzymesubstrate encounter. We previously developed a chemical model for this reaction in which
pyrrole-3-acetate (PAA) was decarboxylated to 3-methyl pyrrole (3MP).1 Reactions
performed at elevated temperatures indicated that the uncatalyzed reaction was extremely
slow (t1/2 = 2.3 x 109 years at pH 10 and 25°C). UroD provided a rate enhancement of 1.2 x
1017 over the uncatalyzed reaction. Catalysis is apparently achieved with the assistance of a
single amino acid side-chain, the guanidinium group of Arg-37. Although Asp-86 apparently
interacts with the nitrogens of all four pyrrole rings of the substrate, it appears to be too
distant from the scissile acetate group to furnish a proton to the incipient methyl product. Arg37 is however in close proximity to the acetate group and may serve as a source of that
proton:
To determine whether guanidine or acetate, or their combination, would exhibit catalysis in
the model reaction, we added guanidine, acetate, separately and together, to 0.1M carbonate
buffer at pH 10 and acetate alone at pH 4.2, in quartz tubes which were heated (130°C to
250°C) and analyzed by 1H NMR.
Guanidinium ion alone, at a concentration of 0.5 M at pH 10 produced a 110-fold increase in
rate, whereas acetate alone, at a concentration of 0.5 M, produced slightly more than a
doubling of the rate. When both were present, the effect of added guanidinium appeared to be
slightly suppressed by the added acetate. Ionic strength increases, tested by addition of KCl,
from 0 to 1M, showed negligible effects on the reaction rates.
Acetate at pH 4.2, where it would be largely protonated, yielded a slightly less than 2-fold
increase in rate, similar to what was observed at pH 10. These data strongly suggest that in
this model system, guanidine catalyzes the decarboxylation of PAA. Likewise in the UroD
reaction, ARG-37 can serve as the catalytic residue facilitating the decarboxylation of Uro III.
This furnishes an unusual example of a single Arg residue providing catalytic assistance in an
extremely difficult decarboxylation reaction.
1
Lewis & Wolfenden 2008, Uroporphyrinogen decarboxylation as a benchmark for the
catalytic proficiency of enzymes, Proc. Natl. Acad. Sci. USA, 105, 17328-17333.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P74
Auxiliary Proteins in Alginate Maturation
Emma K. Farrell†, Krista Arnett†, Howard Robinson‡, Joel T. Weadge§, P. Lynne Howell§,
and Peter A. Tipton†
†
Department of Biochemistry, University of Missouri, Columbia, MO 65211;
Biology Department, Brookhaven National Laboratory, Upton, NY 11973;
§
Program in Molecular Structure and Function, The Hospital for Sick Children, 555
University Avenue, Toronto, Ontario M5G 1X8, Canada;
ⱡ
Department of Biochemistry, University of Toronto
‡
Alginate is a copolymer composed of two epimeric monosaccharide units, mannuronate
and guluronate. It is produced and secreted by the opportunistic pathogen Pseudomonas
aeruginosa, which is responsible for a large proportion of nosocomial infections, and causes
chronic pulmonary infections in patients with cystic fibrosis. Alginate is one of several
factors that cause P. aeruginosa infections to be refractory to treatment. The proteins
required for alginate biosynthesis are encoded by the genes of the alg operon; although each
gene product has been demonstrated to be required for alginate synthesis, the functions of
several of the proteins remain undefined.
The first polymeric species in the alginate biosynthetic pathway is mannuronan, a polymer
of mannuronate residues. C5-Mannuronan epimerase, encoded by the algG gene, catalyzes
the epimerization at C5 in some β-D-mannuronate residues in the polysaccharide to convert
them to α-L-guluronate residues. There is some evidence that the AlgK and AlgX proteins
are associated with C5-mannuronan epimerase in the periplasmic space of the bacteria. We
have found that mannuronan epimerization is inhibited by the presence of AlgX, and that
AlgK does not effect the rate. Product profiles using HPLC indicate that the composition of
the polysaccharide formed in the presence of C5-mannuronan epimerase, AlgK, and AlgX is
different from that formed by C5-mannuronan epimerase alone. The crystal structure of AlgX
reveals that the protein contains a carbohydrate-binding domain and a domain that contains a
catalytic triad whose function remains unknown.
Paradoxically, the alg operon contains algL, which encodes an alginate lyase that
catalyzes degradation of alginate by a β-elimination reaction at C4. Despite the fact that C5mannuronan epimerase catalyzes its reaction via β-elimination and re-formation of the
glycosidic bond between adjacent residues, there is no sequence homology between the
epimerase and the lyase. Kinetic studies have demonstrated that AlgL activity is increased in
the presence of a divalent metal ion, with the most activity being observed with Ca2+. The pH
dependence of the kinetic parameters and the substrate size specificity will be reported. Like
AlgK, AlgX, and AlgG, AlgL is periplasmic, and it may also be a component of a
multiprotein complex involved in alginate maturation. Preincubation of AlgG, K, X, and L
causes a lag in alginate cleavage that is not observed when alginate, AlgG, K, and X are preincubated.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P75
Assessment of Invertebrate Thiaminases by Mass Spectrometry
Ivy Bergquist, Randy L. Sears Jr., Lisa C. Harding, Vatsal M. Patel, Seetha R. Thummuru,
and John V. Schloss
University of New England, College of Pharmacy, Portland, Maine 04103
Invertebrates constitute approximately 60% of the harp seal diet (1). As part of a study to
determine the role of thiamine deficiency in seal strandings, we have developed new mass
spectroscopic methods for assaying thiaminases and determining whether they belong to class
I or to class II. Class I thiaminases catalyze nucleophile-dependent conversion of thiamine to
a thiazole [5-(2-hydroxyethyl-4-methylthiazole] and a nucleophile-linked pyrimidine, while
class II thiaminases catalyze a nucleophile-independent conversion of thiamine to a thiazole
and 2-methyl-4-amino-5-hydroxymethylpyrimidine. We have confirmed that a variety of
mollusks contain class I thiaminases. However, unlike some forms of thiaminase I, the
mollusk enzymes will not utilize glutathione as a substrate. We have also found that some
mollusks also contain a low-pH class II thiaminase, which has not been previously reported.
Dietary thiaminases have been reported to be responsible for sporadic morbidity and mortality
in alligators (2), dogs (3), trout (4), sheep and cattle (5), birds (6), and humans (7).
Supported by a grant from the Gustavus and Louise Pfeiffer Research Foundation
References
1. Hammill et al. (2005) Can J Zool 83:1365-72.
2. Honeyfield et al. (2008) J Wild Dis 44:280-294.
3. Houston and Hulland (1988) Can Vet J 29:383-385.
4. Jaroszewska et al. (2009) Comp Biochem Physiol, Part A 154:255-262;
Thomas (1986) Vet Res Com 10:125-141.
5. Ramos et al. (2005) Can Vet J 46:59-61; Rowe et al. (1980) Trop Anim Prod 5:63-69.
6. Paton et al. (1983) Aus J Zool 31:147-154; Holz et al. (2002) J Avian Med Surg 16:21-25.
7. Nishimune et al. (2000) J Nutr 130:1625-1628;
Lonsdale (2006) Evid Based Complement Alternat Med 3:49-59.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P76
Mechanism-based Affinity Capture of Sirtuins
Jessica Falco, Yana Cen, Dou Yeon Youn, Ping Xu, Anthony A. Sauve*
Department of Pharmacology
Weill Medical College of Cornell University
1300 York Ave
New York, New York 10065
The mammalian sirtuins (SirT1 – 7) are NAD+-dependent protein deacetylases implicated in the
regulation of diverse physiological processes such as insulin secretion, mitochondrial biogenesis,
adipogenesis, stress resistance, DNA-damage responses and tissue survival. The involvement of
these enzymes in these processes has stimulated a tremendous interest in studying them in
biological contexts. The ability to probe for catalytic activities of enzymes and to detect their
abundance in complex biochemical systems has traditionally relied on a combination of kinetic
assays and techniques such as western blots that use expensive reagents such as antibodies. The
ability to simultaneously detect activity and isolate a protein catalyst from such a mixture is even
more difficult and currently impossible in most cases. In this poster we describe an approach that
achieves this goal for sirtuins using novel chemical tools, enabling rapid detection of activity and
isolation of these protein catalysts. Employing an aminooxy-derivatized NAD+ (6-AMX-NAD+)
and a thioacetyl-lysine inhibitor, mechanistic trapping of sirtuins can be accomplished to form
temporally stable complexes that can be crosslinked to an aldehyde-substituted biotin.
Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel
electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight
determination. We show that these tools are cross reactive against a variety of human sirtuin
isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can also react with microbial
derived sirtuins. Finally, we demonstrate with this methodology the ability to simultaneously
detect multiple sirtuin isoforms in reaction mixtures, establishing proof of concept tools for
chemical studies of sirtuins in complex biological samples.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P77
Confirmation of the mechanism of Unsaturated Glucuronyl Hydrolase
Jongkees, SAK and Withers, SG
Department of Chemistry, University of British Columbia
Mammalian glycosaminoglycans are degraded by bacteria through a unique pathway
involving a polysaccharide lyase followed by an unsaturated glucuronyl hydrolase, each with
no mammalian homologues. The first step in this pathway, catalysed by the lyase, has been
determined to proceed via an E1cb mechanism1 and produces an unsaturated sugar at the nonreducing terminus of one of the released products. This unsaturated residue is then removed
by the unsaturated glucuronyl hydrolase, generating a monosaccharide and a new smaller
substrate for the lyase. A novel mechanism has been proposed for this second step based on
crystallographic data,2 but there exist scant other data to support this. Our work aims to
provide confirmation of this mechanistic proposal, in order to allow design of inhibitors of the
second of these two unique bacterial enzymes as potential bacteriostatic agents for prevention
of post-surgical infection.
The unique mechanism proposed for the unsaturated glucuronyl hydrolases involves acid and
possibly base catalysed addition of water across the C4-C5 double bond introduced by the
lyase, followed by non-enzymatic rearrangement of the product hemi-ketal to ultimately break
the anomeric bond and remove this unsaturated terminal residue from the polysaccharide. In
contrast, most polysaccharide hydrolases act by nucleophilic attack of water at C-1, also with
acid/base catalysis, either directly or through a covalent glycosyl-enzyme intermediate. The
key differences to be addressed in discriminating between these two types of mechanisms are
the sites of protonation by the enzyme, being either at C-4 or the leaving group oxygen, and
the site of nucleophilic attack by water, being either at C-5 or at C-1, for the novel proposed
mechanism or a more classical glycoside hydrolase mechanism (respectively).
In order to determine the site of protonation the enzymatic reaction with a synthetic substrate
has been carried out in D2O, and shows signs of being a stereospecific process at C-4.
Similarly, to determine the site of nucleophilic attack the enzymatic reaction with a synthetic
substrate has been carried out in 10% methanol to act as an alternate nucleophile that
generates a stable product, and indicates attack at C-5. Furthermore, several synthetic
substrate analogues have been seen to be processed by the enzyme that would not be expected
to be active with a more classical glycoside hydrolase mechanism, including a thiophenyl
leaving group and a substrate with inverted anomeric stereochemistry.
Results to date are consistent with the novel proposed mechanism, and inconsistent with a
more classical glycoside hydrolase mechanism. Since the activity catalysed is strictly a
hydration rather than a hydrolysis of the substrate, this pathway is operating to give complete
degradation of an alternating polysaccharide to monomers without any glycoside hydrolases.
References
1. Rye, C. and Withers, S. (2002) J. Am. Chem. Soc. 124:9756
2. Itoh, T., Hashimoto, W., Mikami, B. and Murata, K. (2006) J. Biol. Chem. 281:29807
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P78
Design, synthesis and evaluation of new β-lactamase inhibitors
Ronak Tilvawala and R. F. Pratt
Department of Chemistry, Wesleyan University, Middletown, CT 06459
The β-lactam antibiotics are widely used in clinical practice against bacterial infections. The
continuous threat of β–lactamases, however, enzymes that hydrolyse the β–lactam moiety of
these drugs, limits their use. One common strategy for overcoming this problem is
administration of β–lactamase inhibitors along with β–lactam antibiotics. Regrettably, in
recent years, various bacteria have been identified carrying resistance towards the
commercially available β–lactamase inhibitors. Therefore, development of new classes of β–
lactamase inhibitors is urgently needed.
In our laboratory, the O-aryloxycarbonyl
hydroxamates ROCONHOCOOAr have shown considerable reactivity with the active sites of
class A and class C β–lactamases (1). On the basis of this work, several more polar acyl
hydroxamates have been designed and synthesized and kinetics measurements have shown
that these compounds are also irreversible inhibitors of class A and class C β–lactamases.
Their mechanism of inhibition has been investigated.
1. Pelto, R. B. & Pratt, R. F., (2008) Kinetics and Mechanism of Inhibition of a Serine βlactamase by O-Aryloxycarbonyl Hydroxamates, Biochemistry, 47, 12037-12046.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P79
Boronate Inhibitors of Penicillin Binding Proteins: Evaluation in vitro, in
Cell Membranes, and in vivo.
Liudmila Dzhekieva and R. F. Pratt
Department of Chemistry, Wesleyan University, Middletown, CT 06459
Penicillin-binding proteins (PBPs) are bacterial enzymes catalyzing the last step in cell wall
biosynthesis. Recently, a boronate transition state analog inhibitor with Ki of 32 ± 6 nM
against the Actinomadura R39, low molecular weight (LMW) PBP, was developed in our
lab1. This boronate inhibitor mimics the structure of the natural substrate of the PBPs – the
stem peptide of bacterial cell wall. Several new boronate inhibitors of this kind have now
been synthesized and evaluated against the PBPs of E.coli and B. subtilis species in vitro, in
cell membranes and in vivo. A method based on SDS-PAGE and Bocillin FL, a fluorescent βlactam, was developed to quantitatively evaluate reversibly binding inhibitors of PBPs under
each of these conditions.
1. Dzhekieva, Liudmila; Rocaboy, Mathieu; Kerff, Frédéric; Charlier, Paulette; Sauvage, Eric;
Pratt, R. F. Crystal structure of a complex between the Actinomadura R39 DD-peptidase and
a peptidoglycan-mimetic boronate inhibitor: interpretation of a transition state analogue in
terms of catalytic mechanism. Biochemistry 2010, 49, 6411-9.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P80
Human mitochondrial RNA polymerase: Evaluation of the singlenucleotide-addition cycle on synthetic RNA/DNA scaffolds
Eric D. Smidansky, Jamie J. Arnold, Ibrahim M. Moustafa, Shelley L. Reynolds
and Craig E. Cameron
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, PA 16802
The mammalian mitochondrial genome encodes 13 polypeptides and 24 transfer and
ribosomal RNAs essential to the function of the oxidative phosphorylation pathway, which
produces nearly all cellular ATP. The human mitochondrial RNA polymerase (h-mtRNAP) is
responsible for transcribing mitochondrial DNA. Because of the central role of this DNAdependent RNA polymerase in cellular energy production, mutant forms of h-mtRNAP,
prevalent in the human population, may be involved in human disease states. h-mtRNAP is
evolutionarily related to phage T7 RNAP and the two RNAPs are similar structurally based
on homology modeling. We have developed methods for expression of h-mtRNAP to high
levels in E. coli, followed by purification of the protein in large quantities to near
homogeneity. In spite of requiring assistance from at least two accessory proteins to
accomplish transcription in vivo, we find that h-mtRNAP, by itself, rapidly and stably
assembles, via a two-step binding mechanism, on small, synthetic RNA/DNA scaffolds
similar to those known to be utilized efficiently by T7 RNAP1. Assembly on RNA/DNA
scaffolds culminates with h-mtRNAP attaining RNA elongation mode, permitting kinetic
evaluation of single-nucleotide incorporation and pyrophosphorolysis. h-mtRNAP
accomplishes single-nucleotide incorporation relatively slowly. A substantial solvent
deuterium kinetic isotope effect was observed on the rate constant for single-nucleotide
incorporation but not for pyrophosphorolysis, suggesting that chemistry is at least partially
rate limiting for the forward reaction but not for the reverse. Quantitative characterization of
the binding and catalytic kinetics of h-mtRNAP nucleotide incorporation in a minimal in vitro
assay system provides a baseline of information for evaluating the contributions of the
mitochondrial transcription accessory proteins to RNA synthesis. It also permits evaluation of
the biochemical performance of mutant forms of h-mtRNAP that are present in the human
population and that may be linked to disease states. Finally, it is now possible to test the
hypothesis that h-mtRNAP is an accidental and unwanted target for nucleoside analogues that
cause host toxicity when used as antiviral therapeutic agents.
References
1. Temiakov, D., Anikin, M., and McAllister, W.T. (2002) Characterization of T7 RNA
polymerase transcription complexes assembled on nucleic acid scaffolds, J Biol Chem 277,
47035-47043.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P81
Dynamics of Conformational Heterogeneity within the Michaelis Complex
of Lactate Dehydrogenase
Ruel Z. Desamero1, Beining Nie2, Nick Zhadin2, Hua Deng2, and Robert Callender2
1York College of CUNY, Jamaica, NY USA, 2Albert Einstein College of Medicine, Bronx, NY
An enzymatic reaction involves the diffusion-controlled formation of an encounter complex
between the protein and its substrate followed by the appropriate structural and dynamical
arrangements producing Michaelis complex capable of product formation. In forming the
Michaelis complex, the binding pocket is substantially rearranged: protein flaps or loops often
close over the bound ligand, the binding pocket is desolvated, and catalytically important
residues are brought into contact with the substrate. We probed the transient events associated
with the binding of oxamate, a substrate mimic, to lactate dehydrogenase isolated from
Bacillus stearothermophilus (bsLDH) using temperature jump (T-jump) relaxation
techniques. T-jump relaxation monitors the re-equilibration of a chemical system following an
instantaneous increase in temperature induced by a laser pulse tuned to an infrared water
band. The re-equilibration results in changes in the concentration of the species involved, and
the transient changes are characterized using spectroscopic probes. To investigate the
conformational changes associated with the binding of oxamate we studied the LDH from
wild type cells as well as those from various single tryptophan mutants. These mutants were
created by first replacing all tryptophans with tyrosine in wild type bsLDH to create a
tryptophan-less template, followed by reintroduction of a single tryptophan at strategic sites in
the protein. We probed the fluorescence emission of NADH in wild type and mutant bsLDH
to report on the time evolution of the changes within the NADH environment over 100µs to
3ms time scale. Transients collected were then correlated to those resulting from a probe of
tryptophan emissions. The results were then analyzed based on a plausible kinetic model. A
comprehensive picture of the dynamics of ligand binding and Michaelis complex formation in
bsLDH is obtained from the various structural reporters.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P82
An Unprecedented Mechanism in Thiamine-Dependent Enzyme Catalysis:
DXP Synthase
Leighanne A. Brammera, Jessica Motta, Herschel Wadeb, Caren Freel Meyersa
a
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine,
Baltimore, MD, 21205. bDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins
School of Medicine, Baltimore, MD, 21205
Emerging resistance of human pathogens to anti-infective agents make it necessary to develop
new agents to treat infection. The methylerythritol phosphate (MEP) pathway has been
identified as an anti-infective target, as this essential isoprenoid biosynthetic pathway is
widespread in human pathogens, but absent in humans. The first enzyme of the pathway, 1deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the formation of DXP via
condensation of D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate. This thiamine
diphosphate (ThDP)-dependent enzyme bears a novel domain and active site arrangement,
and conflicting reports raise questions about the mechanism of catalysis. DXP synthase has
not been extensively pursued as a drug target due to perceived challenges in selectively
targeting this enzyme. The purpose of this study is to elucidate substrate binding events
toward our long-term goal to develop selective inhibitors of DXP synthase. Here, we present
the results of tryptophan fluorescence binding and kinetic analyses of DXP synthase, and
propose a new model for substrate binding and mechanism. Our results are consistent with a
random sequential mechanism, which is unprecedented in this enzyme class.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P83
Ultra High Resolution Analysis of CTX-M β-Lactamase to study catalysis
and discover novel inhibitors
Derek Nichols, Yu Chen
University of South Florida College of Medicine: Dept of Molecular Medicine, 12901 Bruce
B.Downs Blvd , MDC07 Tampa, FL 33612.
The resistance to second and third-generation cephalosporins due to the production of CTX-M
beta-lactamase from bacteria continues to pose a serious threat to various regions of the
world, particularly in nosocomial settings. The hydrolysis reaction catalyzed by CTX-M
proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a
low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after
attack via a catalytic water, and lastly, the hydrolyzed beta lactam ring product which is
released from the enzyme complex. Structural analysis of CTX-M-9’s X-ray crystallographic
structure at sub-angstrom resolution has enabled us to study enzyme catalysis as well as
perform computational molecular docking. A major current debate is the identity of the
catalytic base involved in deprotonating the nucleophilic Ser70 which attacks the beta lactam
ring. In a recent X-ray crystallographic analysis of CTX-M-9 at 0.88 Å, several key hydrogen
atoms as well as hydrogen bonding states of residues involved in catalysis were identified,
suggesting the catalytic water and Glu166 as the general base. Using this structure as a
template, we screened the ZINC small molecule library with DOCK and identified novel
inhibitors against CTX-M-9. Further structure-based design and synthesis has improved the
affinity of the best inhibitor from 20uM to 2uM. For future studies, we plan to use these noncovalent inhibitors and previously developed boronic-acid covalent inhibitors to study the
hydrogen-bonding network and the proton transfer process at different stages of the reaction
pathway, through ultra-high resolution X-ray crystallography along with neutron diffraction
techniques.
References
1. Chen Y, Bonnet R, Shoicet BK. The Acylation Mechanism of CTX-M β-Lactamase at
0.88 A Resolution. J. AM. CHEM. SOC. (2007) 129: 5378-5380.
2. Chen Y, Shoicet BK. Molecular docking and ligand specificity in fragment-based
inhibitor discovery. Nature Chem. Bio. (2009) 5: 358-364.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P84
Characterization and Lead Development of inhibitors of Dimethylarginine
Dimethylaminohydrolase.
Gayle Burstein , Thomas W. Linsky, Walter Fast
The University of Texas at Austin. 1 University Station A5300 Austin, TX 78712
Nitric oxide (NO) is often described as a double-edged sword.1 On one hand, NO is
essential for cell signaling and host cell defense, and on the other, NO produced by tumor
cells at low levels can actually facilitate the growth and invasiveness of tumor cells.2 Low
level NO production has been found in many malignant human cancers, but not the
surrounding benign tissue.3 The mechanism by which NO participates in tumor biology is not
completely clear, but there is evidence that NO biosynthesis plays a crucial role in
angiogenesis and tumor progression.3 Inhibitors of NO production have already been
suggested as antitumor therapeutics as some of these compounds have been shown to induce
apoptosis and increase susceptibility of melanoma to combination therapy.4 NO is produced
by NO synthase which is inhibited by endogenous asymmetric dimethylarginine (ADMA).
ADMA is in turn hydrolyzed into citrulline and dimethylamine by dimethylarginine
dimethylaminohydrolase (DDAH). Studying the inhibition of DDAH could lead to possible
lead compounds for the development of antitumor therapeutics that block NO production.
Our lab has recently completed a high-throughput screen of fragment-sized
compounds to identify inhibitors of DDAH. The mode of inhibition has been determined for
respresentive hits. Of interest, a structural family of compounds is found to display noncompetitive inhibition. A potential allosteric binding site is indentified in which the
compounds could bind. Several hits from the high-throughput screen are also evaluated in
mammalian cells using an activity-based protein-profiling probe. The use of this probe allows
use rapid screening for the bioavailability and efficacy of the compounds for use in
mammalian cells.
1.
2.
3.
4.
Mocellin, S., Bronte, V. & Nitti, D. Nitric oxide, a double edged sword in cancer biology:
searching for therapeutic opportunities. Med Res Rev 27, 317-352 (2007).
Jenkins, D.C. et. al. Roles of nitric oxide in tumor growth. Proc. Natl. Acad. Sci. U.S.A 92,
4392-4396 (1995).
Fukumura, D., Kashiwagi, S. & Jain, R.K. The role of nitric oxide in tumour progression. Nat.
Rev. Cancer 6, 521-534 (2006).
Ekmekcioglu, S. et. al. Negative Association of Melanoma Differentiation-associated Gene
(mda-7) and Inducible Nitric Oxide Synthase (iNOS) in Human Melanoma: MDA-7 Regulates
iNOS Expression in Melanoma Cells1. Molecular Cancer Therapeutics 2, 9-17 (2003).
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P85
Transition State of Human Nicotinamide Phosphoribosyltransferase
Emmanuel S. Burgos, Mathew J. Vetticatt and Vern L. Schramm
Albert Einstein College of Medicine – Golding G301
1300 Morris Park Avenue – Bronx, NY 10461
For the past decade, Nicotinamide Phosphoribosyltransferase (NAMPT) has been at the center
of interest for biologists and medicinal chemists. Being the rate limiting step of the NAD
salvage pathway, more and more evidence has validated this protein as potential drug target
for most of the concerning health issues of our aging society – cancer, type 2 diabetes and
also a full range of inflammation-related disorders. Because of its critical role in the NAD
metabolic pathway and its intricate relationship with Sirtuin activity (SIRT), modulation of
Nicotinamide Mononucleotide synthesis (NMN, catalyzed very efficiently by NAMPT) has
become a very challenging task for medicinal chemists. Although a few inhibitors have
already been described (e.g. FK866, Ki/Km = 33) and clinic trials are in progress with one
prodrug (GMX1777), actual methodology does not seem to allow discovery of more efficient
compounds. Asymptotic behaviour in this field has challenged us to generate a new class of
more powerful inhibitors by using the Rational Design approach via Transition State (TS)
analysis. Here, we present the TS of HsNAMPT; among the Phosphoribosyltransferase
family, this TS is the first one to be determined and modeled with its natural substrates [NMN
and pyrophosphate (PPi)], also the first to incorporate magnesium atoms in order to generate a
model as close as possible to reality. A milestone for medicinal chemistry, our study will
improve the understanding of this complex enzyme and allow, at term, synthesis of more
powerful drugs against cancer.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P86
15
N Isotope Effects on NH3 for the reaction of NAD+ Synthetase (M.
tuberculosis)
Laurie A. Reinhardt1, Kaiti Chang.2, Barbara Gerratana2, W. Wallace Cleland1
1
Biochemistry Department, University of Wisconsin-Madison, 1710 University Ave., Madison,
Wisconsin 53726. 2Department of Chemistry and Biochemistry, University of Maryland,
College Park Maryland 20742-2010.
NAD+ synthetase (M. tuberculosis) is a multifunctional enzyme belonging to the glutamine
amidotransferase superfamily. Members of this family hydrolyze glutamine to glutamate and
ammonia, and then transfer the ammonia through a molecular tunnel to the
synthetase/synthase domain. In this domain the acceptor substrate carbonyl is activated by
the formation of an acyl-AMP intermediate. Subsequently amidation of the intermediate
occurs by nucleophilic attack by ammonia. In the same fashion, NAD+ synthetase catalyzes
the following transformation of NaAD+ to NAD+.
The X-ray structure of the 6-diazo-5-oxo-L-norleucine-modified enzyme with NaAD+ bound
shows NAD+ synthetase is a homooctomeric enzyme with one active site per monomer, and
an extensive dimer interface. The N-terminal glutaminase domain is like that of the nitrilase
family, and the synthetase domain belongs to the N-type ATP pyrophosphatase family.
Protein sequence alignment of NAD+ synthetase from M. tuberculosis and H. sapiens have
shown differences in their substrate binding interactions. This difference coupled with the
enzyme’s necessity for both replicating and non-replicating microbial cells, make NAD+
synthetase a drug target.
We have proposed to determine the heavy atom kinetic isotope effects for NAD+ synthetase
from both M. tuberculosis and H. sapiens to determine differences in the transition state
structures that can be exploited for the design of a microbial inhibitor. Here, the 15N isotope
effects on this enzymatic reaction in H20 and D2O will be presented.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P87
Computational design of enzymes for novel carbon-carbon bond
formation reactions
Asha Chaubey*, Elizabeth Noey and K.N. Houk
Department of Chemistry and Biochemistry, University of California Los Angeles,
California-90095(USA)
One of the most challenging tasks in computational protein design is to generate active sites
on protein scaffolds capable of catalyzing desired reactions with non-natural substrates. We
have studied two enzymes, namely FBP aldolase, a key enzyme involved in glycolysis as well
as gluconeogenesis in most microorganisms, and LeuA, a key enzyme involved in carboncarbon bond formation in leucine biosynthesis. The main objective of this study is to learn
about the enzyme mechanism and redesign the enzyme to accommodate non-natural
substrates.
To fulfil this objective, we generated models of the key intermediates and transition states in
the reaction pathway with specific residues involved in their respective catalytic reactions by
B3LYP-6-31G(d) quantum mechanical calculations. The initial theozyme structures were then
tested computationally to see if other substrates could undergo the same reaction. Protein
modelling showed how new substrates might be accommodated in the active site to catalyze
non-natural reactions. The non-natural reactions selected in the present study are aldolase
catalysed statin side chain formation, and the acetyl CoA mediated carbon chain elongation
by LeuA to form higher homologs of 2-methyl-1-butanol.
References:
1. L.Jiang, E.A. Althoff. F.R. Clemente, L. Doyle, D. Rothlisberger, A. Zanghellini, J.L.
Betker, F. Tanaka, C.F. Barbas III, D.Hilvert, K.N. Houk, B.L. Stoddard and D.
Baker; De Novo computational design of retro-aldol enzymes; Science,
319(2008)1387-1391.
2. A.K. Samland, G.A. Sprenger; Microbial aldolases as C-C bonding enzymes-unknown
treasures and new developments; Appl. Microbiol. Biotechnol., 71(2006) 253-264.
3. N.S. Blom, Steve Tetreault, R. Coulombe and J. Sygusch; Nature Struct. Biol.
3(1996)856-862.
4. K. Zhang, M.R. Sawaya, David S. Eisenberg and J.C.Liao; Expanding metabolism for
biosynthesis of nonnatural alcohols; PNAS, 105 (2008) 20653-20658.
5. N.Koon, C.J. Squire and E.N.Baker; Crystal structure of LeuA from Mycobacterium
tuberculosis, a key enzyme in leucine biosynthesis; PNAS, 101 (2004) 8295-8300
*On deputation from Indian Institute of Integrative Medicine(CSIR),Canal Road, Jammu-Tawi180001(INDIA)
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P88
Exploring The Inhibitory Effect Of The Hydrazine Functionality On The
Histone Demethylase LSD1
Shonoi A. Barnett, Lindsay Avery, Rong Huang and Philip A. Cole
Affiliation & Address of Author(s) (Johns Hopkins University, School of Medicine,
Department of Pharmacology and Molecular Sciences, 725 N. Wolfe Street, 316 Huntarian,
Baltimore, MD 21205)
Human lysine specific demethylase 1 (LSD1) is a flavin dependent enzyme that oxidatively
removes methyl groups from mono- and di-methylated Lys-4 of histone H3 (Scheme 1).
Previous studies from our laboratory have shown that phenylethylhydrazine is potent suicide
inhibitor with kinact /Ki(inact) = 5.5 x 10-2 µM-1min-1(1). This finding lends itself to suggest that
the hydrazine functionality is attractive for screens of other small molecule inhibitors. To this
end we have embarked on screening small molecule hydrazine derivatives and herein some of
our findings are presented. We find that the compound 3-phenylpropylhydrazine has a kinact
/Ki(inact) = 6.4 x 10-2 µM-1min-1 demonstrating that increasing the chain length by one methyl
group does not increase potency. In addition, substitution of the carbon at position 1 with an
oxygen (2-phenoxyethylhydrazine) does not increase potency but decreases it by ca. 16-fold.
H3-Lys4
H3-Lys4
H3-Lys4
H2 O
LSD1
R
N
H
CH3
R
FAD
H2O2
N
CH2
R
N
CH2OH
FADH2
O2
R = H or CH3
O
H3-Lys4
R
NH2
+
H
H
Scheme 1 Catalytic cycle of flavin mediated amine oxidation.
References
1. Culhane, J. C., Wang, D., Yen, P. M., Cole, P. A. (2010) Comparative Analysis of
Small Molecules and Histone Substrate Analogues as LSD1 Lysine Demethylase
Inhibitors. J. Am. Chem. Soc. 132, 3164-76
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P89
Inhibition of Serine/Amine Amidohydrolases by O-Aryloxycarbonyl
Hydroxamates and Oxathiazol-2-ones: Penicillin acylase and β-lactamases
S. A. Adediran and R. F. Pratt
Department of Chemistry, Wesleyan University, Middletown, CT 06459
Serine β-lactamases have been reported to be irreversibly inhibited by O-aryloxycarbonyl
hydroxamates ArCH2OCONHOCOAr′ (1). The inhibition has been shown to be achieved by
cross-linking of the active site between the nucleophilic serine and an adjacent conserved
lysine. The M. tuberculosis proteasome, an N-terminal protease, has been reported to be
similarly inhibited by oxathiazol-2-ones (2). We here show that O-aryloxycarbonyl
hydroxamates also irreversibly inhibit penicillin acylase, another N-terminal amidohydrolase,
and that the oxathiazol-2-ones inhibit β-lactamases. Structure-activity studies of the former
inhibitors show that the inhibition rate is enhanced by electron-withdrawing aryloxy
substituents and by hydrophobic substitution on the hydroxamate group. These compounds
may be generally effective inhibitors of serine/amine amidohydrolases.
References
1. Pelto R.B. and Pratt, R.F. (2008) Kinetics and Mechanism of Inhibition of a Serine βLactamase by O-Aryloxycarbonyl Hydroxamates. Biochemistry 47, 12037-12046.
2. Lin, G. et al (2009) Inhibitors selective for mycobacterial versus human proteasomes.
Nature 461, 621-628.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P90
Substrate specificity of penicillin-binding proteins
V. V. Nemmara and R. F. Pratt
Department of Chemistry, Wesleyan University, Middletown, CT-06459
Penicillin-binding proteins (PBPs) are enzymes that catalyze the final transpeptidation
reaction of peptidoglycan cross–linking in bacterial cell wall bio-synthesis. β-Lactam
antibiotics restrict bacterial growth by inhibiting these enzymes, which have been studied for
many years. Ironically, the interactions between PBPs and their natural substrates (peptide
moieties in peptidoglycan) are not well understood. Previous studies on the substrate
specificity of PBPs have shown that they often have very little activity against potential
peptide substrates (most of them, however, were not close analogues of peptidoglycan
structure). We have investigated the substrate specificity of low-molecular mass PBPs against
D-alanyl-D-alanine peptides that directly mimic the elements of peptidoglycan structure,
carefully distinguishing carboxypeptidase, endopeptidase and transpeptidase activities.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P91
Coupled pKa Shift Between Conserved Cysteine and Aspartate Plays Key
Role in Protein Splicing
Zhenming Du,1 Yangzhong Liu,2 Chunyu Wang1,*
1
Biology Department, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York 12180; 2Department of Chemistry, University of
Science and Technology of China, Hefei, Anhui, P.R.China 23002
Protein splicing is an autocatalytic process in which an intein excises itself from a precursor
with the concomitant ligation of the two flanking exteins. Protein splicing occurs through a
four-step catalysis but the catalysis roles of key residues are not clearly defined. In inteins,
block F aspartate are conserved and have been shown to play important roles in coordinating
protein splicing but the mechanism is unclear. In this study, we combined NMR
pKa determination with NMR structure determination to study engineered inteins
from Mycobacterium tuberculosis(Mtu) RecA intein. We demonstrate a coupled pKa shift
between the conserved block F aspartate and block A cysteine through a hydrogen bond
(Figure 1). The block F aspartate pKa is elevated by 2 pH units by the block A cysteine and
block A cysteine pKa depressed by 1 pH unit. The pKa shift coupling and NMR structure
suggest that the block F aspartate serves as the hydrogen bond donor and block A cysteine
serves as the hydrogen bond acceptor. This proposed pKa shift coupling promotes
transesterification, a key step that coordinates protein splicing. The proposed mechanism
accounts for the biochemical data supporting the essential role for the block F aspartate.
References
1. Paulus, H. Annu. Rev. Biochem. 2000, 69, 447.
2. Hirata, R.; Ohsumk, Y.; et al. J. Biol. Chem.1990,265,6726
3. Perler, F. B. Nucleic. Acids. Res. 2002, 30, 383.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P92
Enzymatic Mechanism of Protein Arginine Methyltransferase (PRMT)
Probed by Stopped-Flow Fluorescence
You Feng, Y. George Zheng
Department of Chemistry, Georgia State University, PO Box 4098, Atlanta, Georgia 30302.
[email protected]
Molecular mechanisms that mediate epigenetic regulation of gene function include
DNA methylation and histone modifications. Such epigenetic factors control tightly the
normal processes of cellular physiology and organ development. Also it has been widely
demonstrated that epigenetics play important causal roles in human disease occurrence such
as tumorigenesis. Nevertheless, the molecular mechanisms of major histone modifications in
cell biology are very elusive. How these biochemical codes are “written” by chromatin
modifying enzymes and then “read” by downstream effector proteins and protein complexes
to signal a transcriptional status of target genes urgently needs to be elucidated. The focus of
this work is on histone arginine methylation which is catalyzed by protein arginine
methyltransferases (PRMTs). We will report two important new discoveries from our
laboratory on the catalysis of PRMTs. First, we designed fluorescent probes that allow us to
dissect the pre-steady state kinetics of histone arginine methylation. With this fluorescent
approach, we were able to measure the association and dissociation rate constants of enzymesubstrate interaction, as well as the rates of methylation. These data provide significant
insights into the catalytic mechanism of PRMTs. Second, to dissect the interplaying
relationship between different histone modification marks, we investigated how individual
lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in
cis. Our data revealed that the effect of lysine acetylation on arginine methylation depends on
the site of acetylation and the type of methylation. While certain acetylations present a
repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation
generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). These
findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4
acetylation, and unravel that complex intercommunications exist between different PTM
marks in cis.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P93
Global Dynamics in Catalysis by Horse Liver Alcohol Dehydrogenase?
Bryce V. Plapp, Atsushi Yahashiri, Rachel S. Wallace, Karthik K. Shanmuganatham, and
Timothy J. Herdendorf
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA
The involvement of global protein dynamics in catalysis was explored by studying the effects
of substitutions of amino acid residues distal from the active site. Temperature independent
kinetic isotope effects (published studies) provide evidence for quantum mechanical hydrogen
tunneling in this and other dehydrogenases, leading to the suggestion that protein dynamics
are involved in catalysis of hydride transfer. Furthermore, molecular dynamics simulations
suggest that distal residues could affect catalysis through connected networks of amino acids.
If global dynamics contribute to catalysis, we expect that substitutions of conserved amino
acid residues that are distant from the active site, but in hinge regions between domains,
hydrophobic clusters, and other sites that would moderately change protein stability, should
affect catalysis. It is well-established that substitutions of buried residues can destabilize a
protein, by perhaps 1 kcal/mol for each methylene unit removed, as reflected in folding
energetics. Such substitutions have global effects on the cooperative interactions in proteins
and their dynamics. We prepared a variety of singly-substituted enzymes by site-directed
mutagenesis, and determined the steady-state kinetic parameters, transient kinetics for hydride
transfer, and three-dimensional structures at atomic resolution. The substitutions included
G173A, V197I, V207A, I220V, I220L, I220F, V222I, and A317C. The steady-state kinetic
constants (dissociation constants for coenzymes, turnover numbers, and catalytic efficiencies)
for the reactions of NAD+ and benzyl alcohol or NADH and benzaldehyde for the mutated
enzymes were typically within a factor of 2 of those for wild-type enzyme. (In contrast,
substitutions at or near the active site, such as F93A, K228R, I224G, I269S, V292S and
G293A/P295T, cause substantial changes in some kinetic constants, as previously published.)
The apparent rate constants for the transient oxidation of benzyl alcohol (hydride transfer step
as confirmed with deuterated benzyl alcohol) catalyzed by wild-type and mutated enzymes
were also similar, 17 to 30 s–1. Three-dimensional structures (six determined at 1.2 Å) of the
enzymes complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol (mimic of the
Michaelis complex) were essentially identical, except at the site of substitution. Additional
methyl groups squeezed into loose spaces, and removal of methyl groups created cavities that
were not filled with visible water molecules. The added sulfhydryl group in the A317C
enzyme displaced a water molecule. The I220F substitution was accompanied by local
changes in nearby residues. It appears that the enzyme is relatively stable and can tolerate
small substitutions without distorting the overall structure. The lack of structural changes is a
prerequisite for assessing dynamics effects, as structural changes can readily affect activities,
as well as dynamics. Thus, the present results provide no evidence in support of the
hypothesis that global protein motions facilitate catalysis, i.e., the rates of reaction. (These
studies do not provide information about the effects of the substitutions on hydrogen
tunneling, but the contribution of tunneling to the rate of hydrogen transfer may be modest.) It
appears that the structure of the protein scaffold and local interactions and dynamics are
probably most important for catalysis. (Supported by USPHS grant GM078446)
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P94
DNA translocation dynamics revealed by
single molecule nucleotide binding kinetics
Matthew Kellinger1, Jonas Korlach2 and Kenneth A Johnson1
1
Department of Chemistry and Biochemistry, University of Texas, Austin, TX
2
Pacific Biosciences, Menlo Park, CA
Using single molecule methods, we have examined the kinetics of nucleotide binding
to and release from HIV Reverse Transcriptase in complex with DNA terminated by ddT,
d4T, or AZT. The enzyme-DNA complex was absorbed to the surface of zero-mode wave
guides where nucleotide binding was monitored using 5’ fluorescently labeled nucleotides
developed for single-molecule real-time DNA sequencing. Analysis of the pulse-width (PW)
and inter-pulse duration (IPD) lifetimes corresponding to each nucleotide-binding event was
used to determine nucleotide on- and off-rates for the enzyme-DNA complex terminated by
each of the three different analogs. The pulse width and the inter-pulse duration data, defining
the lifetimes of the bound and unbound states, respectively, revealed biphasic kinetics (see
Figures). We describe novel methods for fitting single
Pulse width lifetimes
molecule kinetic data that fully exploit the unique
qualities and high signal:noise ratio of these data. Our
data can be fit to a comprehensive model defining the
kinetics of nucleotide binding, nucleotide-induced
enzyme isomerization, nucleotide release and DNA
translocation. For example, the short-lifetime IPDs
define the second-order rate constant governing
nucleotide binding to the enzyme-DNA complex when
the DNA occupies the post-translocation site (P-site).
The long lifetime IPDs are due to DNA blocking the
nucleotide-binding site (N-site) and thereby reflect the
kinetics governing DNA translocation. Similarly, the
Inter-pulse duration
analysis of the PW distribution defines the kinetics of
nucleotide release and nucleotide-induced enzyme
isomerization leading to a tighter binding state.
We present the first measurement of DNA
translocation dynamics and demonstrate altered
translocation dynamics when DNA is terminated by d4T
or AZT. In particular, retention of the DNA in the
nucleotide-binding site, after incorporation of AZT,
provides a mechanism by which pyrophosphate-mediate
excision can occur on the exposed phosphodiester bond
of the chain terminator, thereby overcoming inhibition.
References:
Kellinger, M. W. and Johnson, K. A. (2010) Nucleotide-dependent conformational change
governs specificity of HIV reverse transcriptase. PNAS 107, 7734-7739.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P95
Allosteric Regulation of α-Isopropylmalate Synthase from Mycobacterium tuberculosis
Ashley K. Casey, Jordyn L. Johnson, and Patrick A. Frantom
Department of Chemistry, The University of Alabama Box 870336 Tuscaloosa, Al 35487
Allosteric regulation of proteins, where the binding of a molecule at one site affects the
chemical properties at a distal site, has been a focus of biochemical studies for over forty
years. In the past ten years, there has been an increase in studies on the role of enzyme
dynamics in allosteric regulation. The enzyme α-isopropylmalate synthase from
Mycobacterium tuberculosis (MtIPMS), which catalyzes the first step in the biosynthesis of
L-leucine, is being studied to obtain a greater understanding of the allosteric regulation and
protein dynamics of a large multidomain enzyme. MtIPMS catalyzes a Claisen condensation
between acetyl-CoA and ketoisovalerate (α-KIV) to form α-isopropylmalate and CoA.
MtIPMS is a homodimer with each monomer consisting of three domains: a regulatory
domain, a linker domain, and catalytic domain. MtIPMS is known to display slow onset
inhibition in the presence of leucine, in which an initial complex is formed quickly followed
by a slower step in which a tighter conformation of the enzyme-inhibitor complex is formed.
The slow onset mechanism is intriguing given that leucine binds over 50 Å from the active
site in the regulatory domain (Figure 1). Based on crystallographic and biophysical
experiments, there is no evidence to suggest a structural change occurs upon leucine binding,
suggesting a mechanism that is based on protein dynamics. Solution phase
hydrogen/deuterium exchange implicated a conserved active site helix that contains ligands
essential to binding the divalent metal ion and α-KIV. This result led to several testable
models of the allosteric mechanism. Site-directed mutagenesis was used to probe the roles of
each residue of the helix in the chemical and regulatory mechanisms. Based on the kinetic
parameters determined for each enzyme variant, each residue participates in the chemical
mechanism. However none of the substitutions had an effect on the kinetics of leucine
inhibition relative to the wild type enzyme. The ability of the divalent metal ion to activate
catalysis in the presence of leucine was also tested. The concentration of divalent cation
necessary for activation was not affected by the presence of leucine. Finally, we investigated
if leucine binding uncoupled the hydrolysis of acetyl-CoA from the condensation reaction.
NMR data shows that the addition of leucine does not uncouple the reaction mechanism. As a
second approach to understanding the allosteric mechanism, alternate amino acids were tested
as inhibitors of the enzyme. Alternate amino acids characterized thus far act as simple
noncompetitive inhibitors with no slow-onset mechanism; inhibition constants range from 200
µM to several millimolar. These results suggest that the determinant for slow-onset inhibition
does not occur in the catalytic domain but rather in the regulatory domain.
Figure 1: Crystal structure of
MtIPMS (PDB id: 3FIG). The
leucine binding site shown in
the regulatory domains binds
50 Å from the active site in
the catalytic domain.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P96
Enzymology as a Tool for Crystallography within the CSGID
Misty L. Kuhn*, Magdalena M. Makowska-Grzyska#, George Minasov*,
Ludmilla Shuvalova*, Ievgeniia Dubrovska*, James Winsor*,
Andrzej Joachimiak#, and Wayne Anderson*
*
Department of Molecular Pharmacology and Biological Chemistry,
Northwestern University Feinberg School of Medicine Chicago, IL
#
Computational Institute University of Chicago, Chicago, IL
The role of the Center for Structural Genomics of Infectious Diseases (CSGID) is to
determine the 3-D structures of proteins involved in emerging and re-emerging infectious
diseases using high-throughput structural biology techniques. One of our goals is to establish
collaborations between our center and the scientific community. We want to combine
expertise of enzymologists, biochemists, biophysical chemists, and structural biologists to
address both structural and functional relationships of proteins. Collaborations may consist of
any of the following: (1) biochemically characterizing a target protein and suggesting possible
ligands for crystallization, (2) providing potential inhibitors for our laboratory to cocrystallize with a specified target, and (3) biochemically characterizing a crystallized protein
and publishing both structural and biochemical data. Additionally, members of the scientific
community may request targets for 3-D structural determination using our pipeline procedure
pending approval from NIAID. They may also request clones and purified proteins free of
charge from our center for their own research, which is ideal for beginning scientists with
limited funding. Recently, our laboratory at Northwestern University has used enzymology as
a tool to aide in protein-ligand complex crystallization efforts of several proteins. These
include a putative glucose-1-phosphate thymidylyltransferase, inosine 5’-monophosphate
dehydrogenases, and a glycerol-3-phosphate dehydrogenase with various ligands.
Optimization of these crystals is currently underway.
This project has been funded in whole or in part with Federal funds from the National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of
Health and Human Services, under Contract No. HHSN272200700058C.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P97
High-Throughput Screening for Selective Inhibitors of N5Carboxyaminoimidazole Ribonucleotide Mutase
Hanumantharao Paritala1, Maria Fawaz1, James B. Thoden2, Hazel M. Holden2 and Steven M.
Firestine1
1. Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and
Health Sciences, Wayne State University, Detroit, MI 48201
2. Department of Biochemistry, University of Wisconsin, Madison, WI 53706
One promising but unexplored area in antimicrobial drug design is de novo purine
biosynthesis. Recent biochemical studies have shown that de novo purine biosynthesis is
different in microbes than in vertebrates. The differences in the pathways are centered on the
synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR). In bacteria, yeast and fungi,
CAIR is synthesized directly from the unstable compound, N5-carboxyaminoimidazole
ribonucleotide (N5-CAIR), by the action of the enzyme N5-CAIR mutase. In contrast,
vertebrates use the enzyme aminoimidazole ribonucleotide (AIR) carboxylase to directly
prepare CAIR from a different intermediate. The biochemical differences between human and
microbial de novo purine biosynthesis provides a rationale for the development of new
antimicrobial agents. Unfortunately, there are no known, selective inhibitors of N5-CAIR
mutase and structural studies indicate that N5-CAIR mutase is nearly identical to AIR
carboxylase. Thus, rational, structure-based drug design is unlikely to be useful in creating
selective inhibitors of N5-CAIR mutase. To remedy this problem, we have conducted highthroughput screening (HTS) against E. coli N5-CAIR mutase using a highly reproducible UV
assay. HTS was done with 48,000 compounds and we found 130 that gave dose-dependent
inhibition of the mutase. A counter screen against the human enzyme revealed that all but 2
compounds also inhibited AIR carboxylase. The remaining two compounds were analyzed
for their mode of inhibition. The results of these studies will be given in the poster.
Acknowledgements: This work was supported in part by funding from the N.I.H. (DK47814
to H.M.H. and GM087467 to S.M.F).
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P98
Long-distance relationships: Communication between the active site lid and
a remote loop in PEPCK.
Sarah M. Holyoak and Todd Holyoak
Department of Biochemistry and Molecular Biology
The University of Kansas Medical Center
3901 Rainbow Blvd., Kansas City, KS 66219
Prior structural studies of the monomeric enzyme phosphoenolpyruvate carboxykinase
(PEPCK) hinted at a relationship between the positions of the active site lid and a remote loop
~ 25 Å away (L153 loop). To test whether the two structures are energetically coupled, we
designed several mutations of the remote loop at leucine 153. In the wild-type enzyme, the
position of L153 shifts from a hydrophobic pocket to a solvent exposed orientation, and in
crystal structures the shift appeared to correlate with the open to closed transition of the active
site lid. The mutations, to aspartate, phenylalanine, and glycine, were designed to increase
polarity, increase hydrophobicity, and increase flexibility, respectively, thus influencing the
position and properties of the L153 loop. Structural and kinetic analyses served to
characterize the effect of the mutations on the loop, as well as any effects on the active site lid
and catalytic function. As predicted, the mutations affected the conformation of the L153
loop, and also perturbed the open-close equilibrium of the active site lid, as was hypothesized.
Surprisingly, mutations in the L153 loop dramatically influenced catalysis, despite the fact
that the site of mutation is ~ 25 Å from the active site. Even more exciting, while mutation to
aspartate or glycine resulted in a kinetically impaired enzyme, mutation to phenylalanine
improved the enzyme, increasing catalytic efficiency (kcat/KM) in the PEP to OAA direction
by an order of magnitude. This increase in catalytic efficiency is due primarily to a decrease
in the KM for GDP. These studies provide evidence for a site in monomeric PEPCK that could
be exploited to allosterically regulate the enzyme due to the energetic coupling of a remote
loop region to the conformational changes necessary for catalysis at the active site.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P99
Site-specific installation of two lysine derivatives in histone H3
Wenshe Liu, Ying Huang and Wei Wan
Department of Chemistry, Texas A&M University, College Station, TX 77843
Tyr
Using an evolved pyrrolysyl-tRNA synthetase- tRNA CUA pair to suppress an ochre mutation
Tyr
and an evolved Methanococcus jannaschii tyrosyl-tRNA synthetase- tRNA CUA pair to suppress
an amber mutation, two different noncanonical amino acids have been incorporated into one
protein in Escherichia coli with high efficiency. Using this strategy, phenylselenocysteine and
Nε-acetyllysine (or Nε-methyllysine) €
can be selectively incorporated into histone H3. The
unique chemical property of phenylselenocysteine in€ histone H3 allows its further
modification to form a lysine derivative so that H3 with two different posttranslational lysine
modifications can be finally obtained. Using this approach to study the interplay between
different H3 lysine modifications will be pursued.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P100
Targeting thymidylate biosynthesis for the development of new antibiotics
Eric M. Koehn1, Scott A. Lesley2, Irimpan I. Mathews3 and Amnon Kohen1
1
Department of Chemistry, University of Iowa, Iowa City, Iowa. 2The Joint
Center for Structural Genomics at the Genomics Institute of Novartis Research
Foundation, San Diego, California. 3Stanford Synchrotron Radiation
Laboratory, Stanford University, Menlo Park, California.
Biosynthesis of DNA depends on activity of the enzyme thymidylate synthase, which
catalyzes the reductive methylation of deoxyuridylate (dUMP) to form the nucleotide
thymidylate (dTMP). Humans and other eukaryotes rely on classical thymidylate synthase
(TSase), whereas many microorganisms and several severe human pathogens produce an
alternative flavin-dependent thymidylate synthase (FDTS). The molecular mechanism of
catalysis differs between classical TSase and FDTS. Perhaps the most notable distinction is
that classical TSase requires an enzymatic nucleophile to covalently bind the substrate and
intermediates during catalysis, however, FDTS enzymes do not.1 Here we show progress
towards further elucidation of the mechanistic details of FDTS enzymes. A variety of
experimental tools including mutagenesis, kinetics, isotopic labeling, and studies using
intermediate analogs further illustrate the differences between TSase and FDTS enzymes. The
findings and chemical mechanism proposed here, together with available structural data,
suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis,
should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis,
its unique mechanism makes it an attractive target for antibiotic drugs.
Acknowledgements: This work was supported by NIH R01 GM065368 and NSF CHE
0715448 to AK and the Iowa CBB and NSF GRFP to EMK.
Reference(s):
1) Eric M. Koehn, Todd Fleischmann, John A. Conrad, Bruce Palfey, Scott A. Lesley,
Irimpan I. Mathews and Amnon Kohen, An unusual mechanism of thymidylate biosynthesis
in organisms containing the thyX gene. Nature 2009, 458, 919-923.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P101
15
N, 13C Primary and 18O Nucleophilic Kinetic Isotope Effects
for the Reaction Catalyzed by Sir2
Mark A. Anderson and W. W. Cleland
Institute for Enzyme Research, Department of Biochemistry, University of WisconsinMadison, 1710 University Avenue, Madison, WI 53726
Brian C. Smith, Dawei Chen, and John M. Denu
Department of Molecular Chemistry, University of Wisconsin-Madison, 553 Medical Sciences
Center, 1300 University Avenue, Madison WI 53706
The silent information regulator 2, (Sir2 or sirtuins) family of proteins are primarily NAD+
dependent histone deacetylases which catalyze the deacetylation of ε-amino-acetylated lysine
residues from protein substrates releasing the products O-acetyl-ADP-ribose (OAADPr),
nicotinamide, and deacetylated protein. It is proposed that the carbonyl oxygen of the acetyl
group attacks C-1 of ribose to displace nicotinamide. We have determined the primary kinetic
isotope effects (KIE) for N-1 of nicotinamide during the initial step of catalysis using the
acetyl (1.0115 ± 0.0015), hydroxyacetyl (1.0281 ± 0.0011), and propionyl (1.0305 ± 0.0016)
peptides. The propionyl analog has the highest KIE which we consider to be very close to the
intrinsic isotope effect. The primary carbon effect will be determined for each analog by
oxidation of the residual ribose with bromine/periodate to produce CO2 from the C-1 position.
Adenosine 5’-diphosphoribose pyrophosphatase (ADPr-PPase) has been overexpressed and
purified and, used in conjunction with alkaline phosphatase (AP), allows for the isolation of
ribose. The nucleophilic KIE will be determined for the attacking carbonyl oxygen by the
remote label method. 12C-1, 16O sodium propionate and 13C-1,18O sodium propionate have
been synthesized, mixed to natural abundance levels, and incorporated into an 11-mer peptide.
After the enzymatic reaction is complete, the propionate will be cleaved from the propionyl
ADP-ribose product, isolated by azeotropic distillation with octane, and degraded to CO2 for
determination of the 13C mass ratio at C-1 of the propionate. The results of these experiments
will allow us to determine the nature of the transition state of the reaction.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P102
Functional Assignment of the N245 Residue of 4-Hydroxyphenylpyruvate
Dioxygenase from Streptomyces avermitilis: A Key Residue in
Hydroxylation Regiospecificity
June Brownlee, Dhara Shah, Judith Bates and Graham R. Moran
Department of Chemistry and Biochemistry, The University of Wisconsin- Milwaukee.
3210 N. Cramer st, Milwaukee, WI 53211.
4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the first committed step of tyrosine
catabolism. This ancient pathway has been adapted to a variety of purposes unique to each
kingdom of life. As such, the inhibition of HPPD has far reaching consequences and is the
basis for herbicides, therapeutics for specific metabolic disorders and the suppression of
bacterial virulence. HPPD catalyses a rather unusual reaction involving substrate
decarboxylation, aromatic hydroxylation and substituent migration. HPPD and one other
enzyme, hydroxymandelate synthase (HMS) catalyze dioxygenation reactions involving the
same two substrates, 4-hydroxyphenylpyruvate (HPP) and molecular oxygen. However HMS
does not catalyze aromatic hydroxylation or the substituent migration. Instead HMS simply
hydroxylates the benzylic carbon forming hydroxymandelate. HPPD and HMS have the same
fold and share many active residues, exhibiting a limited number of distinct active site
residues. We have been concerned with elucidation of the catalytic mechanism HPPD and
understanding the structural differences that result in either HMS or HPPD activities. Site
directed mutants of the residues unique to one or the other activity have revealed the
importance of asparagine 245 in HPPD that is an isoleucine in HMS. Our data indicate that
this residue is directly involved in both the positioning of the substrate for hydroxylation of
the aromatic ring and the orchestration that results in the 1,2-migration of the aceto side chain
on the ring to form homegentisate. Product analysis studies of variants at this position show a
variety of products can be induced to form. A serine residue in this position induces the
formation of quinolacetic acid a product in which ring hydroxylation has occurred but the
shift has not. Still other mutants form hydroxyphenylacetic acid, a product resulting from
uncoupling of the hydroxylation reaction. The majority of mutations at this position, however,
make multiple products and indicate a bifurcation of the reaction coordinate at the
hydroxylation step. Changes in the ratio of these products in the presence of per-deutero-HPP
provides a simple, accurate means to measure kinetic isotope effects on the hydroxylation step
and there bye unveil the nature of the first intermediate following hydroxylation(1).
References
1.
Panay, A. J., and Fitzpatrick, P. F. (2008) Kinetic isotope effects on aromatic and
benzylic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase as
probes of chemical mechanism and reactivity, Biochemistry 47, 11118-11124.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P103
Small-molecule inhibitor of a human deubiquitylating enzyme complex
Junjun Chen1, Thomas Dexheimer2, Yongxing Ai1, Anton Simeonov2, and Zhihao Zhuang1
1
Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware,
Newark, DE, 19716; 2 NIH Chemical Genomics Center, National Human Genome Research
Institute, National Institutes of Health, Bethesda, Maryland, United States of America
Deubiquitylating enzymes (DUB) antagonize the activities of the ubiquitin ligases by
removing ubiquitin moiety from a target protein or from a ubiquitin chain. Although the
function of most human DUBs remains to be determined, it has become clear that the DUB
activities are indispensible for the normal functions of a number of cellular processes.
Abnormal cellular expression of DUBs or the loss of function due to mutation in certain DUB
genes have been linked to various human diseases. Among the five DUB families, ubiquitinspecific proteases (USP) are emerging as promising targets for pharmacological intervention
because of their connection to many human diseases, including prostate, colon and breast
cancer, pediatric acute lymphoblastic leukemia, and familial cylindromatosis. Despite the fact
that more than sixty USPs have been identified in humans and that many are implicated in
various human diseases, few USP inhibitors were reported. In this work, we identified highly
specific small-molecule inhibitors against a human deubiquitinating enzyme complex through
high-throughput screening. Mechanistic and inhibition studies revealed that the inhibitor acts
noncompetitively with a submicromolar Ki. We further demonstrated that treating human
cisplatin-resistant cancer cells with the USP inhibitor sensitizes the cancer cells to the
platinum drug. Through a quantitative drug interaction analysis, we demonstrated that this
inhibitor acts synergistically with cisplatin in inhibiting the proliferation of cancer cells
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P104
Insights into the chemical mechanism of eukaryotic UDP-galactopyranose
mutases
Oppenheimer, Michelle, Qi, Jun, Llanos-Valezquez, Janice, and Sobrado, Pablo.
Virginia Tech, Department of Biochemistry, Blacksburg, VA 24061
UDP-galactopyranose mutase (UGM) is a flavin-dependent enzyme that catalyzes the
conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf).
UGM is the sole biosynthetic source of Galf, which is found on the cell surface of many
pathogens where it plays a key role in pathogen-host interactions. Deletion studies show that
UGM is essential in Mycobacterium tuberculosis and is important for virulence in Aspergillus
fumigatus and Leishmania major. Since UGM is not found in mammals, it is considered an
ideal drug target. Understanding the mechanism of UGM will aid in the development of
highly specific inhibitors. Previous studies suggest two potential mechanisms one in which
the required reduced flavin acts as a nucleophile attacking the anomeric carbon (C1) of
galactose (Figure 1A), the second involves electron transfer by the flavin (Figure 1B). Our
studies focus on understanding the mechanism of the eukaryotic UGMs from A. fumigatus
and Trypanosoma cruzi. Rapid-reaction kinetic analysis of reduced UGM titrated with UDPGalp indicate the formation of the putative iminium ion, as shown by characteristic decrease
in absorbance at 410 nm and an increase at 500 nm. This intermediate was trapped by
incubating reduced UGM with UDP-Galp and sodium cyanoborohydride and isolated by
HPLC. Conformational changes appeared to occur during catalysis as shown by a protection
from proteolysis by trypsin upon reduction and further protection upon addition of substrate.
Also our studies suggest half-site reactivity in the tetrameric A. fumigatus UGM as it is
capable of stabilizing half of the bound flavin in the reduced active form.
Figure 1. Two proposed mechanisms for UDP-Galactopyranose Mutase.
This work was supported by grants from the National Institute of Health (GM 094469) to P.S. and by a
predoctoral fellowship from the American Heart Association (10PRE4160020) to M.O
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P105
Substrate Identification for the HDAC orthologue Rpd3 using in vivo
photo-crosslinking
Noah Wolfson,1 Jody Lancia, 2 Anna Mapp, 2,3 Carol Fierke1, 2, 3
Dept. of Biological Chemistry,1 Chemical Biology,2 and Chemistry,3 University of Michigan,
Ann Arbor MI 48109
The histone deacetylase (HDAC) family is comprised of 19 proteins that catalyze the selective
deacetylation of ε-acetyl lysines. Acetylation has been shown to alter the activity of proteins
with aberrant acetylation implicated in diseases such as cancer and Alzheimer’s disease, and
identification of each HDAC’s substrates would aid in understanding of mechanisms involved
in these diseases [1]. To date, the primary means for identification of HDAC substrates has
been through the use of pull down experiments [2-4]. We are developing a method that will
be better able to identify transient interactions between the HDACs and their binding partners.
Using nonsense suppression technology, a non-natural amino acid, p-benzoyl-Lphenylalanine (pBpa) is site-specifically incorporated into the substrate binding face of RPD3
[5], a yeast HDAC homologue [1]. Upon irradiation, pBpa crosslinks with proximal proteins,
allowing for their identification [6]. Using this method, pBpa has been incorporated into
various positions of the Rpd3 substrate binding surface and multiple RPD3-protein crosslinks
have been observed in the presence and absence of RPD3 inhibitors. We are working on
identifying the crosslinked molecules by mass spectrometry.
Acknowledgements: This work was supported in part by funding from the N.I.H. (R01
GM40602-22).
References:
1.
Yang, X.J. and E. Seto, 2008, "The Rpd3/Hda1 family of lysine deacetylases: from
bacteria and yeast to mice and men." Nat Rev Mol Cell Biol. 9(3): p. 206-18.
2.
Kovacs, J.J., et al., 2005, "HDAC6 regulates Hsp90 acetylation and chaperonedependent activation of glucocorticoid receptor." Mol Cell. 18(5): p. 601-7.
3.
Vaziri, H., et al., 2001, "hSIR2(SIRT1) functions as an NAD-dependent p53
deacetylase." Cell. 107(2): p. 149-59.
4.
Lee, H., et al., 2006, "Histone deacetylase 8 safeguards the human ever-shorter
telomeres 1B (hEST1B) protein from ubiquitin-mediated degradation." Mol Cell Biol.
26(14): p. 5259-69.
5.
Chin, J.W., et al., 2003, "An expanded eukaryotic genetic code." Science. 301(5635):
p. 964-7.
6.
Dorman, G. and G.D. Prestwich, 1994, "Benzophenone photophores in biochemistry."
Biochemistry. 33(19): p. 5661-73.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P106
Kinetically Controlled Allosteric Activation:
Potassium creates a ball and socket joint in IMP Dehdyrogenase
Thomas V. Riera1, Lianqing Zheng2, Helen R. Josephine3, Donghong Min2,
Wei Yang2,4 * and Lizbeth Hedstrom3,5*
Graduate Program in 1Biochemistry and Departments of 3Biology and 5Chemistry, Brandeis
University, 415 South St., MS 009, Waltham, MA 02454 USA;
2
Institute of Molecular Biophysics and 4Department of Chemistry and Biochemistry and,
Florida State University, Tallahassee, FL 32306 USA.
Allosteric activators usually shift the equilibrium distribution of enzyme conformations to
favor a catalytically productive structure; the rate of interconversion between conformations is
generally believed to be rapid and therefore inconsequential. Several observations suggested
that the usual allosteric mechanism does not apply to the activation of IMP dehydrogenase
(IMPDH) by monovalent cations. We have determined the kinetic mechanism of
Cryptosporidium parvum IMPDH in the absence of monovalent cations. Surprisingly, neither
substrate affinity nor the rates of the chemical transformations were K+-dependent. Instead,
the presence of K+ accelerates a conformational change required for completion of the
catalytic cycle by at least a factor of 65. We performed alchemical free energy simulations
using the orthogonal space random walk strategy to computationally analyze how K+
promotes this conformational change. The simulations recapitulate the preference of IMPDH
for K+, validating the computational models and providing insight into the structural basis of
monovalent cation selectivity. When K+ is replaced with a dummy ion, the residues of the
monovalent cation binding site relax into stable secondary structure. K+ mobilizes this region,
facilitating conformational exchange. These experiments suggest that K+ activation is
predominantly a kinetic process and that K+ acts as a ball and socket joint to facilitate the
interchange of protein conformations. We propose that IMPDH represents a new paradigm of
kinetically controlled allosteric regulation that may be widespread.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P107
The Human Hotdog-fold Protein THEM4 is Both a Protein Kinase Bα
(Akt1) Regulator and a Fatty Acyl-CoA Thioesterase
Hong Zhao, Anthony Choudhry, Jeffery D. Carson, Michael Schaber, Peter J. Tummino,
Lusong Luo, Marco Bossoffi and Debra Dunaway-Mariano
Enzymology and Mechanistic Pharmacology, Oncology CEDD, GlaxoSmithkline,
Collegeville, PA 19426, USA; Department of Chemistry and Chemical Biology, University of
New Mexico, Albuquerque, NM 87131, USA; Department of Biochemistry and Molecular
Biology, University of New Mexico Health Sciences, Albuquerque, NM 87131
The serine/threonine kinase protein kinase B (PKB), also known as Akt, plays important roles
in cell growth, metabolism, proliferation and survival. Activated PKB/Akt catalyzes the
phosphorylation of an array of kinases that participate in signal transduction and is itself
regulated by phosphorylation, and by interaction with nonkinase protein partners. Published
in vivo studies suggested that the apoptotic effect of the carboxyl-terminal modulator protein
CTMP (also known as hTHEM4) is mediated through its interaction with the PKB isoform
PKBα/Akt1 (1, 2). We have previously shown that hTHEM4 is a medium-long chain fatty
acyl-CoA thioesterase (3). In the present work we employed in vitro methods to demonstrate
that hTHEM4 binds activated PKBα/Akt1 and that it inhibits PKBα/Akt1 kinase activity. In
addition, hTHEM4 was also shown to impair PKBα/Akt1 phosphorylation by the kinases
PDK1 and mTORC2. Finally, PKBα/Akt1 was found not to significantly inhibit hTHEM4
thioesterase activity. A model for the binding interaction of PKBα/Akt1 with the N-terminal
domain of hTHEM4 is proposed.
References
1. Maira, S. M., Galetic, I., Brazil, D. P., Kaech, S., Ingley, E., Thelen, M., and Hemmings, B.
A. (2001) Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt
and v-Akt at the plasma membrane, Science 294, 374-380.
2. Parcellier A, Tintignac LA, Zhuravleva E, Cron P, Schenk S, Bozulic L, Hemmings BA.
(2009) Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that
sensitizes cells to apoptosis. Cell Signal 21, 639-50.
3. Zhao H, Martin BM, Bisoffi M, Dunaway-Mariano D. (2009) The Akt C-terminal
modulator protein is an acyl-CoA thioesterase of the Hotdog-Fold family. Biochemistry 48,
5507-5509.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P108
Insights Into the Divergence of Function in the Hotdog-fold Protein
Products of Gene Duplicates II: A Case Study of the E. coli Hotdog
Thioesterases YbdB (EntH) and YdiI
John Latham, Rui Wu, Danqi Chen, Karen Allen, and Debra Dunaway-Mariano
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque,
NM and Department of Chemistry, Boston University, Boston, MA
Bacterial thioesterases perform a variety of biological functions that serve secondary
metabolic/catabolic pathways and primary lipid pathways. The vast majority of these enzymes
have evolved within the hotdog fold enzyme family. The E. coli genome encodes seven
hotdog thioesterases, which include ybdB (aka EntH) and ydiI, which display high (59 %)
sequence identity. Here we show through biochemical and X-ray structural analyses that these
two thioesterases possess nearly indistinguishable three-dimensional structures and similar
mechanisms of catalysis, yet they display distinct substrate specificity profiles. Based on our
previous work (1), we know that ybdB specializes in the release of an acyl/aroyl unit from
mischarged EntBs of the enterobactin biosynthetic pathway. Based on YdiI’s comparatively
wide bacterial range, high activity towards aroyl-CoA thioesters, and co-location of its
encoding its gene with a gene encoding a FAD-binding PCMH-type domain containing
oxidoreductase, we propose that ydiI functions in a novel aromatic xenobiotic pathway.
References
1. Chen, D., Wu, R., Bryan, T. L., and Dunaway-Mariano, D. (2009) In vitro kinetic analysis
of substrate specificity in enterobactin biosynthetic lower pathway enzymes provides insight
into the biochemical function of the hot dog-fold thioesterase EntH. Biochemistry 48, 511513.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P109
Biotin synthase: How iron-sulfur clusters
are used to make carbon-sulfur bonds
Andrew M. Taylor, Christine E. Farrar, Michael R. Reyda, Corey J. Fugate,
and Joseph T. Jarrett
Department of Chemistry, University of Hawaii, Honolulu, HI 96822
Biotin synthase (BS) is an iron-sulfur enzyme that catalyzes the formation of the
thiophane ring of the essential vitamin biotin. The enzyme contains two FeS clusters: a
[Fe4S4]2+/+ cluster is bound to a consensus CxxxCxxC sequence motif that is characteristic of
S-adenosylmethionine (SAM or AdoMet) radical enzymes, while a [Fe2S2]2+ cluster is bound
deep within a β barrel at the other end of the active site. In common with other AdoMet
radical enzymes, BS uses the [Fe4S4] cluster to transfer one electron into the sulfonium of
AdoMet, resulting in formation of methionine and a highly reactive 5´-deoxyadenosyl radical.
This radical can oxidize dethiobiotin by abstracting a hydrogen atom from either the C9
methyl or the C6 methylene positions, generating a transient dethiobiotinyl carbon radical.
We propose a stepwise mechanism in which the C9 position is preferentially oxidized and
then quenched by the µ-sulfide of the nearby [Fe2S2] cluster. Consistent with this mechanism,
the 9-mercaptodethiobiotin is generated as a stable monothiolated intermediate that is then
consumed as the C6 position is oxidized and the thiophane ring closed. Formation of new C-S
bonds is also accompanied by reduction of the remnant [Fe2S2] cluster, generating an EPRdetectable paramagnetic intermediate best described as a [Fe2S(RS)]+ cluster. Differential 34S
labeling of the [Fe2S2] and [Fe4S4] clusters confirms that the thiophane sulfur initially
originates from the [Fe2S2] cluster and that additional slower turnovers can occur utilizing S2–
from the buffer. In vivo turnover likely requires assistance from iron-sulfur cluster assembly
systems (Isc or Suf systems). Finally, we have used the tight-binding inhibitor S-adenosyl-Lhomocysteine to provide evidence that the enzyme is half-site active, and that only one active
site within the dimeric enzyme participates in both initial and subsequent turnovers. Several
recently discovered AdoMet radical enzymes catalyze formation of C-S bonds at unactivated
carbon positions, and our mechanistic results are likely to be broadly applicable to this new
class of enzymes.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P110
Thermodynamic Profiles for Tight Binding Inhibitors to MTANs
Keisha Thomas, Vern L. Schramm
Department of Biochemistry, Albert Einstein College of Medicine, Bronx NY 10461
The bacterial enzyme, methylthioadenosine nucleosidase (MTAN) is a homodimeric enzyme
which is involved in autoinducer (AI-1 and AI-2) quorum sensing pathways and adenine
recycling. MTANs are potential targets for antibiotic design since they are not present in
humans.
Salmonella enterica and Vibrio cholerae MTANs have 95% and 60% sequence homology
respectively with Eschericia coli MTANs but have different Km values for substrates and tight
binding inhibitors.
The differences in binding affinities are explored by binding thermodynamic profiles using
isothermal calorimetry.
MT-DADMe-ImmA and pCl-PhT-DADMe-ImmA are tight binding inhibitors of these
MTANs. Inhibitor binding thermodynamic profiles for S. pneumoniae, S. enterica and V.
cholerae vary.
MTAN
S.
pneumoniae
S.
enterica
V.
cholerae
E.
coli
Table 1
MT‐DADMe‐ImmA
Ki
(pM)
24,000
44
74
2
pCl‐PhT‐DADMe‐ImmA
Ki
(pM)
360
44
74
0.047
Table 1 shows the Ki
values for the various
MTANs and the inhibitorsMT-DADMe-ImmA and
PCl-PhT-DADMe-ImmA
Figures 1 and 2 show
the thermodynamic
parameters for binding
of the MTANs to MTDADMe-ImmA and
pCl-PhT-DADMeImmA respectively.
The data was fit using
a two-site model on
Origin 7.0. All ITC
experiments were
carried out at 25 0C.
Figure1
Figure 2
References
1.
Singh, V., Evans, G.B., Lenz, D.H., Mason, J.M., Clinch, K., Mee, S., Painter, G.F.,
Tyler, P.C., Furneaux, R.H., Howell, P.L., and Schramm, V.L. (2005) Femtomolar
transition state analogue inhibitors of 5'-methylthioadenosine/S-adenosylhomocysteine
nucleosidase from Escherichia coli. J. Biol. Chem. 280, 18265-18273.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P111
Replica Path Determination of Bortezomib Deboronation
Joseph D. Larkin*, Peng Tao*, H. Lee Woodcock, Milan Hodoscek, and Bernard R. Brooks*
*The National Heart, Lung, and Blood Institute, The National Institutes of Health
Recently, we reported a quantum chemical investigation of the oxidative deboronation of
boroglycine, a model compound of the chemotherapeutic bortezomib. Our PBE1PBE/6311++G** (MP2/cc-pVTZ) results show a possible oxidative reaction mechanism for the
enzyme-catalyzed metabolism of this novel drug. Herein, we present results from a QM/MM
investigation of the oxidative metabolism of both the model compound boroglycine and
bortezomib using the Replica Path method. Results show good agreement with the
experimental identification of metabolites indicating oxidative deboronation to be the
principal route of metabolism.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P112
Biosynthesis of 1-Deoxynojirimycin
Lorraine Clark & Nicole Horenstein
Department of Chemistry, University of Florida, Gainesville, FL 32611
The azasugar 1-deoxynojirimycin (DNJ) is a glucose derivative in which the ring oxygen is
substituted with a nitrogen atom. Natural sources of DNJ include microbes, such as Bacilli
and Streptomyces species, as well as various plants, such as Morus alba (Mulberry) and the
dayflower Commelina communis. As potent inhibitors of glycosidases, DNJ and analogs have
been used in Asian folk medicine for centuries and nowadays function as important medicinal
compounds for the treatment of diabetes and Gaucher’s disease. Labeling studies in the early
1990s established glucose as a precursor of DNJ in Bacilli and Streptomyces species and have
led to a proposed biosynthetic pathway. However, the exact biosynthetic pathway of DNJ has
not been elucidated in any species to date, despite its biologically relevant role.
A genome search of the known DNJ producer Bacillus amyloliquefaciens was performed in
order to identify possible gene candidates. For this search, the results of the labeling studies
were considered as well as genes with potential roles in carbohydrate metabolism. A cluster of
three genes was identified, referred to as gabT1, yktc1, and gutB1, which exhibit putative
transaminase, fructose phosphatase and dehydrogenase functions, respectively. By combining
the results of the labeling studies along with the discovery of this gene cluster, a possible
biosynthetic pathway of DNJ in Bacillus amyloliquefaciens was developed, shown in the
following scheme:
!
We present kinetic! and spectroscopic data confirming loss of DNJ production in a gabT1
B. amyloliquefaciens knockout. Furthermore, complementation studies have narrowed down
potential GabT1 amino donors candidates to either asparagine or N-acetylornithine. Current
experiments focus on cloning the enzymes required to measure GabT1 activity via coupled
assays as well as carrying out these assays using spectroscopic techniques.
References
1. Inouye, S.; Tsuruoka, T.; Niida, T. J. Antibiot., Ser. A. 1966, 19, 288-292.
2. Hardick, D. J.; Hutchinson, D. W.; Trew, S. J.; Wellington, E. M. H. Tetrahedron,
1992, 48, 6285-6296.
3. Hardick, D. J.; Hutchinson, D. W. Tetrahedron, 1993, 49, 6707-6716.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P113
Detailed Kinetic Analysis of Irreversible EGFR Inhibitors as a Method of
Driving Drug Design.
Phillip A. Schwartz and Brion W. Murray
Pfizer Global Research and Development, La Jolla Laboratories, San Diego, CA
Activating mutations in a variety of receptor tyrosine kinases (RTKs) can lead to uncontrolled
cell proliferation and have been identified as the cause of numerous carcinoma. Such
mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) lead to
aberrant signaling and are responsible for a large subpopulation of tumors. EGFR can be
activated through distinct molecular mechanisms, including point mutations in the activating
loop (A-loop) or in-frame deletions adjacent to the N-terminal regulatory αC-helix, both
effecting the catalytic domain conformation.
The activated EGFR mutants are initially responsive to inhibition/inactivation by small
molecule tyrosine kinase inhibitors, but a second active site mutation results in acquired
resistance, drastically lowering the efficacy of these compounds. Resistance involves the
addition of a second mutation at Thr-790 (T790M), a gatekeeper residue at the entrance to the
hydrophobic portion of the ATP binding pocket
Tight-binding irreversible inhibitors of EGFR and EGFR mutants have been characterized by
numerical integration of a series of progress curves at varying inhibitor concentrations using
global analysis software (DynaFit). The results are interpreted in terms of the kinetic
constants that govern binding and reactivity and are used to drive the optimization of
increasingly more potent compounds with greater selectivity for EGFR mutants.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P114
Investigation of Substrate Recognition in the Yeast Candida albicans
Protein Prenyltransferases
Elaina A. Zverina1 and Carol A. Fierke2
Chemical Biology Doctoral Program1 and
Departments of Chemistry and Biological Chemistry2
University of Michigan, Ann Arbor, MI
Infections by opportunistic fungal pathogens have become a major clinical problem in the past
decades due to the increasing number of immunocompromised patients who are highly susceptible to
such infections, namely individuals infected with the human immunodeficiency virus (HIV) and AIDS
patients, organ and bone marrow transplant recipients under immunosuppressive therapy, or cancer
patients treated with cytotoxic drugs. The most important fungal pathogens are Candida albicans and
other yeasts of the genus Candida, Cryptococcus neoformans, and the mold Aspergillus fumigatus.
Different classes of antimycotic drugs are available to treat fungal infections, but the pathogens can
develop resistance to these antifungal drugs either by drug-specific mechanisms or by overexpression
of efflux pumps which confers resistance to many structurally and functionally unrelated compounds.
This clearly illustrates the need for novel antifungal agents that would not be subject to existing
resistance mechanisms.
The protein prenylation pathway may be a potential new target for antifungal treatment.
Prenylation is an essential post-translational lipid modification that leads to membrane localization of
many signaling and regulatory proteins. Specifically, my research focuses on C. albicans protein
farnesyltransferase (FTase), which adds a 15-carbon isoprenoid moiety and protein
geranylgeranyltransferase-I (GGTase-I) which adds an analogous 20-carbon isoprenoid unit. In each
case, the lipid is transferred to the sulfur of the cysteine residue of the substrate four amino acids
upsteam of the C-terminus, and the Cxxx tetrapeptide sequence confers recognition elements.
In order to characterize C. albicans prenyltransferases and determine their molecular
recognition determinants, C. albicans FTase and GGTase-I enzymes were overexpressed, purified and
kinetically characterized with known substrates. A screen and characterization of 350 peptides has
been carried out with C. abicans GGTase-I. These peptides were carefully selected to represent a
diverse set of Cxxx combinations, as well as potential in vivo substrates obtained from available C.
albicans genomic sequences in TrEMBL database. This information will shed light on substrate
recognition by C. albicans prenyltransferases, identify potential in vivo substrates, and comparison
between mammalian and C. albicans substrate and non-substrate pools will allow for a better
assessment of FTase and GGTase-I as targets for antifungal therapies.
References
1.
Morschhäuser J. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet Biol. 2010 47(2):94‐106 2.
Hast MA and Beese LS. Structure of protein geranylgeranyltransferase‐I from the human pathogen Candida albicans complexed with a lipid substrate. J Biol Chem 2008 283(46):31933‐40 3.
Hougland JL, Hicks KA, Hartman HL, Kelly RA, Watt TJ, Fierke CA. Identification of novel peptide substrates for protein farnesyltransferase reveals two substrate classes with distinct sequence selectivities. J Mol Biol. 2010 395(1):176‐90 22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P115
Role of dynamics in tuning fidelity of RNA-dependent RNA polymerase
elucidated by molecular dynamics simulation
Ibrahim M. Moustafa1, Coray M. Colina2, Craig E. Cameron1
1
Department of Biochemistry and Molecular Biology, 2Department of Materials Science and
Engineering, The Pennsylvania State University, University Park, PA 16802, USA
The viral RNA-dependent RNA polymerase (RdRp) is absolutely essential for replication of
all RNA viruses.
During RNA synthesis, RdRp makes one or two nucleotide
misincroporations in each replication cycle which result in population diversity of the virus.
This population diversity contributes to the ability of the RNA virus to infect the host. The
physical basis underlying polymerase fidelity is not clear but suggested to be linked to
dynamics of the enzyme [1]. By using molecular dynamics (MD) simulation of two mutants
of picornaviral RdRp from poliovirus (PV) that exhibit altered fidelity compared to the wildtype (WT) enzyme, we show that dynamics play a role in polymerase fidelity. We explored
dynamics of the WT PV RdRp, the higher-fidelity mutant (G64S), and the lower-fidelity
mutant (H273R) by performing 25 ns all-atom MD simulations. Analysis of MD simulations
revealed that the single site mutations affect the global protein dynamics and the correlations
between observed motions that involve functional elements. The changes in the pattern of
dynamics of the WT enzyme caused by single amino acid substitutions are intriguing
knowing that this pattern was shown in our previous work to be highly conserved across
RdRps from four members of picornaviruses, including PV, even when primary sequences
were only 30% identical. Interestingly, differences in dynamics between WT PV RdRp and
its mutants are more pronounced than differences in dynamics observed between various
picornaviral RdRps. Notably, in contrast to the observed differences in dynamics of the G64S
and H273R mutants compared to the WT enzyme, crystal structures of the WT PV RdRp and
its fidelity variants are almost identical (RMSD ~0.3 Å). We propose that changes in
dynamics contribute to the observed variation of polymerase fidelity. This new information
can be exploited for development of antiviral therapeutics and rational design of vaccine.
References:
1‐ Cameron, C. E., Moustafa, I. M. & Arnold, J. J. (2009). Dynamics: the missing link
between structure and function of the viral RNA-dependent RNA polymerase? Curr
Opin Struct Biol 19, 768-74
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P116
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P117
Real-time Fluorescence Polarization Assay for Steady-state Kinetics and
Screening for Inhibitors of Bacterial RNase P
Xin Liu1, Carol Fierke1, 2
1
Department of Chemistry, and 2Department of Biological Chemistry, University of Michigan,
Ann Arbor MI 48109
New antibiotic targets and treatments are in urgent need due to the continued emergence of
drug-resistant bacteria [1]. Ribonuclease P (RNase P) is an enzyme that catalyzes the
maturation of the 5’ termini of precursor-tRNAs (pre-tRNAs). Bacterial RNase P is an
attractive potential antibacterial target because it is essential for cell survival and has distinct
structural differences from its eukaryotic counterpart [2]. We have developed a real-time
fluorescence polarization (FP) assay using pre-tRNA labeled with a fluorophore at the 5’end
to screen for inhibitors of bacterial RNase P. This FP assay is amenable to both kinetic
studies and endpoint measurements and has been used to measure inhibition of B. subtilis
RNase P by neomycin B (IC50 = 23 ± 6 µM) and kanamycin B (IC50 = 95 ± 30 µM).
Furthermore, a primary screen for RNase P inhibitors in a 266 compound library in a 96-well
plate format was carried out. This assay provides a Z’ factor of 0.8 and is rapid, cost-effective
and robust. We are optimizing the assay for a 384-well microplate format to perform highthroughput screening of larger compound libraries. The high-throughput methodology for
screening for RNase P inhibitors will allow both a test of the hypothesis that RNase P is a
viable drug target and investigation of molecular recognition of inhibitors by this enzyme.
Acknowledgements: We thank Lyra Chang and Professor Jason Gestwicki for help with
setting up the compound library, Professor Anna Mapp for sharing the TECAN plate reader,
and Elaina Zverina and Dr. John Hsieh for helpful discussions. This work is supported by
funding from the National Institutes of Health (GM 55387).
References:
1.
Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL. 2007, “Drugs for bad bugs:
confronting the challenges of antibacterial discovery.” Nat. Rev. Drug Discov. 6: pp.
29-40.
2.
Eder PS, Hatfield C, Vioque A, Gopalan V. 2003, “Bacterial RNase P as a potential
target for novel anti-infectives.” Curr. Opin. Investig. Drugs. 4: pp. 937-943.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P118
A Critical Pleiotropic Interaction Governing the Specificity of
5-epi-aristolocholene Synthase and its Evolutionary Implications
Troy Wymore1 and Charles L Brooks III2
1
National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center, 300
South Craig Street, Pittsburgh, PA 15213
2
Department of Chemistry and Biophysics, University of Michigan, 930 North University
Avenue, Ann Arbor, Michigan 48109
The attainment of new catalytic functions from an existing protein fold is a major
force guiding evolutionary change but one that is perhaps only beginning to be understood.
Understanding the evolution of enzymatic function at the physicochemical level requires first
that probable evolutionary paths that interconvert an enzyme’s specific function from one to
another through accessible mutational changes be discovered and thus a functional landscape
be defined. Through a landmark study of sesquiterpene synthases in which a 5-epiaristolocholene synthase was transformed to a premnaspirodiene synthase through mutational
swaps of nine residues (none of which make specific contacts with the substrate) as well as
experimental classification of all 512 proteins with different combinations of these nine
residues a functional landscape underlying the evolution of sesquiterpene chemical diversity
was revealed [O’Maille et al, Nature Chemical Biology, 2008]. Also of significance, the
catalytic cycle of both synthases passes through several common intermediate states and only
diverges in the last few chemical reactions. Because of all these interesting properties,
elucidating the mechanistic basis for this evolution of new function has broad implications for
a more complete understanding of chemical allostery, functional epistasis, evolvability and
protein design concepts.
In this presentation, we demonstrate through hybrid Quantum Chemical/Molecular
Mechanical (QC/MM) simulations that a pleiotropic intermolecular interaction between a
tyrosine residue (which we discovered was highly conserved in plant sesquiterpene synthases
through a large-scale, high-resolution multiple sequence alignment) and the eudesmane
carbocation intermediate governs the specificity of Nicotiana tobaccum 5-epi-aristolocholene
synthase. This intermolecular interaction raises the barrier for methylene transfer (the path to
the premnsapirodiene product), lowers the barrier for methyl transfer and results in an
intermediate positioned for the final proton transfer mediated by the enzyme to give 5-epiaristolocholene. Our current working hypothesis is the nine outer-sphere residues that
interconvert the function of the two synthases act to alter the structure and/or dynamics of this
functional element (Arg441-Asp444-Tyr520-Asp525) with respect to the common eudesmane
carbocation intermediate.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P119
Pre-steady state analysis of trans-3-chloroacrylic acid dehalogenase
Jamison P Huddleston, Gottfried K. Schroeder, Kenneth A. Johnson, Christian P. Whitman
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, and
Division of Medicinal Chemistry,Colllege of Pharmacy, University of Texas, Austin, TX,
78712
trans-3-Chloroacrylic acid dehalogenase (CaaD) is a heterohexamer in the tautomerase
superfamily. CaaD catalyzes the breakdown of trans-3-haloacrylic acids (chloro- and bromo-)
to malonate semialdehyde. Malonate semialdehyde is further degraded to acetaldehyde which
is utilized in the Kreb’s cycle. To help identify the determinants of substrate specificity and
understand the chemical mechanism, fluorescent CaaD mutant(s), having minimal kinetic
differences from wild-type CaaD (wt CaaD), were constructed. With these mutant(s), presteady state kinetics of trans-3-bromoacrylic acid dehalogenation can be monitored using
stopped flow fluorescence. As wt CaaD does not contain tryptophan residues, a non-critical
residue near the active site, α-Y60, was changed to α-W60. The steady state parameters were
determined for wt CaaD and the α-Y60W mutant using trans-3-bromoacrylic acid (UV
assay). The Km value for the CaaD mutant decreases about 1.5-fold and kcat is the same, within
experimental error. To further assess the kinetic effect of the mutation, rapid quench
experiments were performed for both the wild-type and α-Y60W mutant. Both enzymes
show burst kinetics suggesting that product release is at least partially rate limiting. The burst
rate for wild-type is only about 3-fold faster than that of the α-Y60W mutant. Given the
minimal kinetic differences observed between wt and mutant, stopped flow fluorescence
experiments were carried out. The results suggest CaaD and trans-3-bromoacrylic acid come
to a rapid initial equilibrium followed by a slow chemical step and rate limiting product
release. Using Kintek Global Explorer Pro, six experiments were simultaneously fit by
simulation using a minimal 4-step kinetic model, with initial estimates for the rate constants
determined by conventional data analysis. The resultant global fits and the associated
individual rate constants account for the experimental data. Further stopped flow experiments
can now be performed with this mutant to test proposed roles for critical residues. Another
superfamily member, cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has been similarly
analyzed. Steady state values (Km and kcat) indicate that CaaD and cis-CaaD are kinetically
similar in processing their respective 3-haloacrylic acid isomers. However, a comparison of
the CaaD and cis-CaaD analyses suggests possible differences in their mechanisms. Both
CaaD and cis-CaaD show rate limiting product release, but stopped flow experiments suggest
substrate binding, chemistry, and product inhibition are not identical. The differences are
being examined further.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P120
The rate-limiting step for the second-order hydrolysis of many substrates
by acetylcholinesterase (kcat/KM) is gated entry to the catalytic triad
Jeffrey T. Auletta, Veena Beri, Robert Chapman, Juanita F. Wood, Abdul H. Fauq and
Terrone L. Rosenberry
Mayo Clinic College of Medicine, Departments of Neuroscience and Pharmacology,
Jacksonville, FL 32224
Catalytic hydrolysis of the neurotransmitter acetylcholine by the enzyme acetylcholineesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant kcat/KM that
approaches 108 M-1s-1. AChE contains a narrow and deep active site gorge with two sites of
ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the
gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to
contribute to catalytic efficiency with the chromogenic acetylcholine analog acetylthiocholine
(AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the
A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the
P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site
to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs
acetylhomothiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine)
and acetylnorthiocholine (Ac-nSCh, which deletes one methylene group from thiocholine).
We synthesized Ac-hSCh and Ac-nSCh and compared their catalytic parameters with AChE
to those of AcSCh. Values kcat/KM for Ac-hSCh and Ac-nSCh were 2% of that for AcSCh,
while values of kcat for Ac-hSCh and Ac-nSCh were 30% and 5% of that for AcSCh,
respectively. We note that kcat/KM appeared to show greater specificity for AcSCh than kcat.
The kcat/KM of nearly 108 M-1s-1 for AcSCh is close to the theoretical diffusion-controlled
limit for the substrate association rate constant, and kinetic solvent isotope effects are in
agreement with this interpretation. The value of kcat for AcSCh is reduced by a factor of 2.5
in D2O, consistent with a rate limiting deacylation step that involves proton transfer. In
contrast, the value of kcat/KM is decreased by only a factor of 1.1 in D2O, a value too low to
indicate rate-limiting proton transfer and suggestive of rate-limiting substrate association. For
comparison, a slowly hydrolyzed acetanilide substrate, 3-(acetamido)-N,N,N-trimethylanilinium, clearly equilibrates with the A-site, and its kcat/KM is decreased by a factor of 2.1 in
D2O. We report here that the D2O isotope effects on kcat/KM for Ac-hSCh and Ac-nSCh also
approach the 1.1 value obtained for AcSCh, even though the kcat/KM values for these analogs
are much too low to involve rate-limiting substrate association. We propose that kcat/KM for
these substrates is rate-limited by the unimolecular movement of bound substrate from the
P-site to the A-site. Such gating has been proposed previously1 but only with regard to
substrate bulk, with smaller substrates gated more efficiently. Since Ac-nSCh is smaller than
AcSCh but still very inefficiently gated, we assert that P-site dynamics have evolved to
provide highly selective gating and thus high specificity precisely for the acetylcholine
structure.
1
Zhou, H. X.; Wlodek, S. T.; & McCammon, J. A. (1998). Proc. Natl. Acad. Sci. U. S. A. 95, 9280-83
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P121
Enzyme Diversity and Evolution among the Enolase Superfamily:
Understanding Sequence/Function/Structure Relationship
Ayano Sakai,1 Alexander A. Fedorov,2 Elena V. Fedorov,2
Steven C. Almo,2 and John A. Gerlt1
1
Departments of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign,
Urbana, IL 61801,
2
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461
The exponential growth in the volume of data from genomic DNA sequences produces
a growing gap between the number of known protein sequences and the number of proteins
which have an accurately assigned function. Despite our effort, there still remains the
problem of assigning correct functions for numerous proteins. This assignment of
physiological function to proteins is a major challenge in genomic biology at this day. Due to
the relatively small number of protein folds in contrast to the ever-growing and diverse
sequence database, we have focused on (β/α)8-barrel fold, particularly the Enolase
Superfamily, one of the most ubiquitous folds in the Protein Data Bank (PDB) for function
assignment. This investigation also elucidates interesting insights for understanding the
structural basis for catalysis as well as the design principles that can be used to develop
catalysts for new reactions.
The Enolase Superfamily, whose members have diverse sequences but make up
enzyme catalytic domains of close homology, is a good target to understand
sequence/function/structure relationship. Members of the Enolase Superfamily share a
common structural motif, the (β/α)8 barrel, a conserved catalytic site, and a catalytic
intermediate stabilized by a divalent metal ion. These shared features provide an excellent
opportunity to investigate enzyme evolution. However, we have faced a difficulty in that
there are more members in the Enolase Superfamily that do not follow our present
classification from the sequence profile and we have realized that the Superfamily is more
convoluted than we imagined.
Here, we examine one of our first targets in the Enolase Superfamily, an enzyme from
Agrobacterium tumefaciens (PDB: 1rvk). The enzyme is capable of performing D-glucarate
dehydration yet its active site architecture is different from an authentic E. coli D-glucarate
dehydratase (PDB: 1jdf), which was also experimentally and structurally characterized in our
lab. In this example, the two active sites performing the same function are compared to
examine their substrate binding manners as well as their chemistry for catalysis in order to
elucidate the enzyme evolution and catalytic principles.
Exploring members of the Enolase Superfamily whose functions have not yet been
assigned will offer new insights into enzyme evolution. Our investigation on these new,
diverse proteins in the Enolase Superfamily will provide us with a clue to lead a deeper
understanding of evolutionary relationship among protein sequence, structure, and function.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P122
QueE, a New Member of the Radical SAM Superfamily, is Involved in the
Biosynthesis of 7-deazapurines
Reid M. McCarty, Vahe Bandarian
University of Arizona. Department of Chemistry and Biochemistry
1041 E. Lowell St. Tucson, Arizona 85721
Pyrrolopyrimidine containing compounds, collectively referred to as 7-deazapurines,
comprise a broad range of nucleoside antibiotics produced by various species of Streptomyces
bacteria. In addition, a 7-deazapurine moiety is found in the hypermodified tRNA base
archeosine, located in the D-loop of archaeal tRNA, and queuosine, located in the wobble
position of select tRNAs in almost all organisms. We previously discovered the pathway for
the biosynthesis of 7-deazapurines in which GTP is utilized as starting material. The pathway
is composed of three enzymatic steps. The third step, a complex heterocyclic rearrangement
yielding the 7-deazapurine base, is catalyzed by QueE, a new member of the radical SAM
superfamily of enzymes. We are currently investigating the properties and catalytic
mechanism of QueE in the hope of expanding our knowledge of the repertoire of chemistry
achieved by this fascinating class of enzymes.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P123
Evidence for a phosphorylated enzyme intermediate in the reaction
catalyzed by methylthioribose-1-phosphate isomerase
Vamsee Veeramachineni and Andrew S. Murkin
Department of Chemistry, The State University of New York at Buffalo, Buffalo, NY 14260
The methionine salvage pathway ensures the recovery of reduced sulfur from 5′methylthioadenosine, a byproduct of polyamine biosynthesis. The first step universal to all
organisms is the isomerase-catalyzed conversion of 5-methylthioribose 1-phosphate (MTR1P)
to 5-methylthioribulose 1-phosphate (MTRu1P). Whereas other aldose-ketose isomerizations
proceed through the aldose’s aldehyde form, MTR1P exists as a phosphate ester, and
therefore, MTR1P isomerase must utilize a unique mechanism. [32P]MTR1P was incubated
with the isomerase in a 10:1 molar ratio, and the enzyme was denatured with 6 M urea. Gel
filtration chromatography on BioGel-P2 was monitored for protein by UV absorbance at 280
nm and for radioactivity by scintillation counting. Protein eluted in early fractions, coincident
with low levels (ca. 103 cpm) of 32P radioactivity, prior to elution of the bulk (ca. 105 cpm) of
radioactivity. We propose a mechanism involving covalent catalysis via a phosphorylated
enzyme adduct, generating 5-methylthioribose as an intermediate. Further investigations using
[methyl-3H3]MTR1P and polyacrylamide gel electrophoresis are underway.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P124
Reactions of Propiolic Acid with Tautomerase Superfamily Dehalogenases:
Analysis and Implications
Gottfried K. Schroeder, William H. Johnson, Jr., Jamison P. Huddleston,
Kenneth A. Johnson, and Christian P. Whitman
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology,
and Division of Medicinal Chemistry,
College of Pharmacy, University of Texas, Austin, TX 78712
cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) is a member of the tautomerase
superfamily, exhibiting the characteristic β-α-β fold and a catalytic amino-terminal proline.
This enzyme converts the cis-isomer of 3-chloroacrylic acid (1) to malonate semialdehyde (2)
and HCl. cis-CaaD also accepts another substrate, 2,3-butadienoic acid (3), catalyzing its
conversion to acetoacetate (4), with similar kinetic parameters. Recent experiments have
indicated that the hydration of 2,3-butadienoic acid (3) proceeds via a Schiff base mechanism
utilizing Pro1. Given the reactivity of malonate semialdehyde (2), it could act as a product
inhibitor by forming a Schiff base at the active site. To this end, we sought a chemical
synthesis (halide free) of the product malonate semialdehyde (2). Experiments utilizing the
starting material propiolic acid (5) under mildly basic conditions at elevated temperature (70
°C) proved successful (~70% yield by NMR). Further tests showed that this compound is
also processed to malonate semialdehyde (2) by cis-CaaD and two other superfamily
members: CgX and trans-3-chloroacrylic acid dehalogenase (CaaD). Preliminary stoppedflow fluorescence experiments with cis-CaaD and propiolic acid (5) were also conducted.
The results suggested that the initial step for cis-CaaD catalyzed propiolic acid (5) hydration
is slower than that of cis-3-chloroacrylic acid (1).
The closely related cis-CaaD homologue CgX also processes propiolic acid (5), albeit at a
faster rate (≥ 10-fold) than cis-CaaD. Moreover, CgX catalyzes the rapid decarboxylation of
malonate semialdehyde (2) to acetaldehyde (6). This decarboxylase activity is typically found
as a discreet activity in another enzyme of the 1,3 dichloropropene catabolism pathway,
malonate semiadehyde decarboxylase (MSAD), which shares little overall sequence identity
with CgX. It may also be of note that while the active sites of CgX and cis-CaaD are
essentially identical, cis-CaaD does not exhibit any detectable decarboxylase activity.
Together, these findings may have broader implications for the bacterial processing of
acetylene compounds and the promiscuity of the β-α-β fold.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P125
Structural evidence that peroxiredoxin catalytic power is based on
transition state stabilization
Leslie B. Poole1, Andrea Hall2, Derek Parsonage1 and P. Andrew Karplus2
1
Department of Biochemistry, Wake Forest University Health Sciences, Medical Center Blvd.,
Winston-Salem NC 27157
2
Departments of Biochemistry and Biophysics, Oregon State University,
Corvallis OR 97331
Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection
and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The
Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cellwide peroxide reduction, but the origins of their high activity have been mysterious. We
present here a novel structure of human PrxV at 1.45 Å resolution that has a dithiothreitol
bound in the active site with its diol-moiety mimicking the two oxygens of a peroxide
substrate. This suggests diols and similar di-oxygen compounds as a novel class of
competitive inhibitors for the Prxs. Common features of this and other structures containing
peroxide, peroxide-mimicking ligands or peroxide-mimicking water molecules reveal
hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen
track along which the peroxide oxygens move as the reaction proceeds. Key insights include
how the active site microenvironment activates both the peroxidatic cysteine side chain and
the peroxide substrate, and is exquisitely well-suited to stabilize the transition state of the inline SN2 substitution reaction that is peroxidation. *Supported by a grant from the NIH to
L.B.P. with a subcontract to P.A.K. (RO1 GM050389).
Figure: Cartoon of interactions in the Prx active-site and locations of active site water
molecules in unliganded, fully folded structures of Prx proteins (with bound H2O2 in green).
References
Hall A, Parsonage D, Poole LB, and Karplus PA. J Mol Biol 402: 194-209, 2010
Hall A, Nelson K, Poole L, and Karplus PA. Structure-based insights into the catalytic power
and conformational dexterity of peroxiredoxins. Antioxid Redox Signal. Epub Oct 24, 2010
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P126
Selective Inhibition of Ras Function in Live Cells using Caged
Farnesyltransferase Inhibitors
Daniel Abate-Pella,1 Nicholette A. Zeliadt,2 Timothy M. Dore,3 David A. Blank,1 Elizabeth V.
Wattenberg,2 and Mark D. Distefano1
1
Departments of Chemistry and Medicinal Chemistry, University of Minnesota
Minneapolis, MN 55455
2
Division of Environmental Health Sciences, University of Minnesota
Minneapolis, MN 55455
3
Department of Chemistry, University of Georgia
Athens, GA 30602
The creation of caged molecules involves the attachment of protecting groups to biologically
active compounds such as ligands, substrates, and drugs that can be removed under specific
conditions. Photoremovable caging groups are the most common due to their ability to be
removed with high spatial and temporal resolution. Such molecules have been used in a
plethora of biochemical studies ranging from probing the mechanisms of enzymatic reactions
to controlling cellular activity. Ras proteins are small GTP-binding proteins that belong to a
superfamily of regulatory polypeptides that participate in key signal transduction pathways
and regulate such diverse cellular phenomena as nuclear transport, cytoskeletal structure and
cell division. For proper function, Ras proteins must be post-translationally modifed by the
enzyme protein farnesyltransferase (PFTase). Because of this requirement, the growth of
cancer cells that signal via oncogenic forms of Ras can be arrested by inhibiting PFTase; as a
result, a large number of PFTase inhibitors have been developed including several that are
available commercially including FTI, 1. Hence, in principle, caged inhibitors of PFTase
could be used to modulate Ras function. To design such compounds, we reasoned that
functionalization of the thiol group present in 1 would greatly reduce the affinity of the
compound for PFTase. For the caging moiety, we elected to explore the utility of thioetherlinked BHQ and Bhc groups. While these have not previously been employed for sulfur
protection, they were chosen in lieu of others because they have both been shown to be useful
for both one- and two-photon uncaging processes. Here, we first describe the synthesis and
photochemistry of two caged inhibitors of PFTase, BHQ-FTI (2) and Bhc-FTI (3). We then
show that these molecules can be photolyzed with UV light to release FTI (1) that inhibits Ras
farnesylation (observed via Western blot analysis), Ras membrane localization (detected by
confocal microscopy), and downstream signaling (fibroblast morphology). Finally, we show
that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to
inhibit Ras localization.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P127
Investigation of the HAD Superfamily Phosphatidic Acid Phosphatase
Human Lipin1 and its HAD Superfamily Phosphatase Regulator Dullard
Jeremiah Farelli, Rui Wu, Megan Garland, Li Zheng, Debra Dunaway-Mariano and Karen
Allen
Department of Chemistry, Boston University, Boston MA and Department of Chemistry and
Chemical Biology, University of New Mexico, Albuquerque, NM
Two human haloalkanoate dehalogenase superfamily (HADSF) phosphatases, lipin1 and
dullard, function in cellular lipid metabolism and membrane biogenesis through the highly
regulated conversion of phosphatidic acids (PA) to diacylglycerides (1). Lipin1 catalyzed PA
dephosphorylation is mediated by the C-terminal C0 type HADSF phosphatase domain,
whereas lipin’s cellular location/protein partners and hence biological function, is directed by
phosphorylation/dephosphorylation events, which occur at numerous sites located primarily
on the lipin N-terminal domain. The C0 type HADSF phosphatase domain of dullard
catalyzes lipin dephosphorylation and thus functions in the regulation of lipin1 (2). Dullard is
an ER membrane protein bound by via an N-terminal domain transmembrane helix, whereas
lipin1 is a large soluble protein found in the cytoplasm and the nucleus. As the first step
towards defining the structural determinants of lipin1/dullard substrate recognition, catalysis
and regulation, each enzyme has been successfully produced as a soluble construct in E. coli
and expressed in native form in transfected human HEK293 cells. The progress made towards
structure-function analysis will be reported.
References
1. Phan, J., and Reue, K. (2005) Lipin, a lipodystrophy and obesity gene, Cell Metab 1, 73-83.
2. Kim, Y., Gentry, M. S., Harris, T. E., Wiley, S. E., Lawrence, J. C., Jr., and Dixon, J. E.
(2007) A conserved phosphatase cascade that regulates nuclear membrane biogenesis, Proc
Natl Acad Sci U S A 104, 6596-6601.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P128
Insights Into the Divergence of Function in the Hotdog-fold Protein
Products of Gene Duplicates II: A Case Study of the Human Hotdog
Thioesterases hTHEM5 and hTHEM4
Hong Zhao, Dennis Dominguez, Kap Lam, Osnat Herzberg and Debra Dunaway-Mariano
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque,
NM and the University of Maryland, College Park, MD
Human membrane biogenesis and lipid metabolism require the participation of fatty acyl-CoA
thioesterases, which have evolved within two protein fold families, one of which is the hotdog
fold family. Despite links to numerous fatal human diseases, little is known about the
biochemical and biological functions of most thioesterases. Here we examine structurefunction relationships in two paralogous thioesterases of the hotdog family, namely hTHEM5
and the tumor suppressor hTHEM4 (aka Akt1Carboxy-Terminal Modulator Protein) (1, 2).
The production of hTHEM5 and hTHEM4 in E. coli facilitated in-vitro biochemical studies,
leading to substrate specificity profile and catalytic mechanism determination, as well as to Xray crystallographic structure determination. We found that hTHEM5 and hTHEM4 share
37% sequence identity, similar three-dimensional structures and nearly identical catalytic
sites. The substrate specificity profiles measured for hTHEM5 and hTHEM4 are however,
different, as are hTHEM5 and hTHEM4 cellular locations. We show by in-vivo GFP
imagining and by antibody/western blot analysis that hTHEM5 is a mitochondrial matrix
thioesterase in contrast to hTHEM4, which is reported to localize in the mitochondrial inner
membrane space to be released into the cytoplasm upon mitochondrial stress (3). Whereas
hTHEM4 functions in cell apoptosis (2, 3), we propose that hTHEM5 functions in
mitochondrial fatty acid synthesis, with the likely physiological substrate being the
mitochondrial FAS fatty acyl-ACP.
References
1. Knobbe, C. B., Reifenberger, J., Blaschke, B., and Reifenberger, G. (2004)
Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator
protein gene in glioblastomas, J Natl Cancer Inst 96, 483-486.
2. Maira, S. M., Galetic, I., Brazil, D. P., Kaech, S., Ingley, E., Thelen, M., and Hemmings, B.
A. (2001) Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt
and v-Akt at the plasma membrane, Science 294, 374-380.
3. Parcellier A, Tintignac LA, Zhuravleva E, Cron P, Schenk S, Bozulic L, Hemmings BA.
(2009) Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that
sensitizes cells to apoptosis. Cell Signal 21, 639-50.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P129
Identification and Structure Determination of Novel Adenine Deaminase
Enzymes
Alissa M. Goble, § Zhening Zhang¤, Subramanyam Swaminathan,¤ and Frank M. Raushel§
§
Department of Chemistry, Texas A&M University, College Station TX
¤
Biology Department, Brookhaven National Laboratory, Upton, NY
Three previously uncharacterized proteins, Pa0148, AAur_1117, and Patl2390 have been
annotated as adenosine deaminases. The genes encoding these proteins were cloned from
Pseudomonas aeruginosa PAO1, Arthrobacter aurescens TC1, and Pseudomonas atlantica
T6c and the proteins expressed and purified. It has been determined that these proteins do
not catalyze the deamination of adenosine. Pa0148 and Aaur_1117 will efficiently catalyze
the deamination of adenine to produce hypoxanthine with a kcat/KM of 1.4 x 106 M-1s-1 and 3.1
x105 M-1s-1, respectively. In addition, both enzymes will catalyze the dechlorination of 6chloropurine. Aaur_1117 will also accept 6-methoxypurine, N6-methyladenine and 2,6diaminopurine as substrates. Despite a 45% sequence identity with Pa0148 and AAur_1117,
Patl2390 does not catalyze the deamination of adenine. Substrates for Patl2390 include N6methyladenine, N6-dimethyladenine, zeatin, N6-isopentenyladenine, thiomethylpurine, and 6chloropurine. The X-ray structure of Pa0148 was determined at 2.5 Å resolution (Protein
Data Bank entry 3OU8). This protein folds into a distorted (β/α)8 barrel with a single zinc ion
that is characteristic of members of the amidohydrolase superfamily. The amidohydrolase
superfamily is comprised of 24 clusters of orthologous groups of proteins (COG) and these
three proteins belong to COG1816. All members of COG1816 were previously annotated as
adenosine deaminase enzymes. Based on sequence alignments and ligand binding
interactions in the active site of Pa0148, conserved active site residues have been used to
identify 116 other proteins that are predicted to deaminate adenine.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P130
A Study on the Transesterification of Methyl Aryl Phosphorothioates in
Methanol Promoted by Cd(II), Mn(II) and a Synthetic Pd(II) Complex
David R. Edwards, Alexei Neverov and R. Stan Brown
Department of Chemistry, Queen’s University, Kingston, Ontario, Canada, K7L 3N6
Phosphorothioate esters are analogues of phosphate esters in which one or more of the
non-bridging oxygen atoms have been substituted with sulfur. Thio effects, defined as the
ratio of rate constants for reaction of a phosphate ester relative to that of the corresponding
phosphorothioate ester (kO/kS), have been employed as mechanistic probes for enzyme
catalyzed phosphoryl transfer reactions to deduce transition state structure and sites of metal
ion coordination.1 The current study was undertaken in an effort to better understand the
metal-ion/phosphorothioate interactions that result in the often times large thio effects (kO/kS
up to ~108)2 observed for metalloenzyme catalyzed phosphoryl transfer.
Methanol solutions containing Cd(II), Mn(II) and a synthetic Pd(II) complex are
shown to promote the methanolytic trans-esterification of O-methyl O-4-nitrophenyl
phosphorothioate at 25 oC with impressive rate accelerations of 106-1011 over the background
methoxide promoted reaction. A detailed mechanistic investigation of the methanolytic
cleavage reaction catalyzed by the Pd(II) complex was undertaken. A sigmoidal shaped plot
of log(kcat) vs ss pH displays a broad ss pH independent region from 5.6 ≤ ss pH ≤ ~10 with a
kminimum = (1.45 ± 0.24) x 10-2 s-1) and a [lyoxide] dependent wing plateauing above a
kinetically determined ss pK a of 12.71 ± 0.17 to give a kmaximum = 7.1 ±1.7 s-1. Brønsted plots
were constructed for reaction of O-methyl O-aryl phosphorothioates at ss pH 8.7 and 14.1,
!
!
!
corresponding to reaction in the mid-points of the low and high ss pH plateaus. The Brønsted
coefficients !
(βLG) are computed as -0.01 ± 0.03 and -0.86 ± 0.004 at low and high ss pH ,
respectively. Possible reaction mechanisms are discussed. Significantly
this catalytic system
!
is also shown to promote the methanolytic cleavage of O,O-dimethyl phosphorothioate
producing (CD3O)2P=O(S-) with a half time for reaction!of 34 minutes.
!
References
1. Lassila, J. K.; Herschlag, D. Biochemistry, 2008, 47, 12853.
2. Zhao, L.; Liu, Y.; Bruzik, K. S.; Tsai, M. –D. J. Am. Chem. Soc. 2003, 125, 22.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P131
Impact of temperature on the time required for primordial chemistry and
enzyme evolution
(Supported by NIH Grant GM-18325)
Randy B. Stockbridge, Charles A. Lewis, Jr., Yuan Yang and Richard Wolfenden
Dept of Biochemistry and Biophysics, Univ. of North Carolina, Chapel Hill, NC
There has been controversy about whether life originated in a hot or cold environment, and about whether
enough time has elapsed for life in general, and enzymes in particular to have evolved to their present
levels of complexity.
All reactions are accelerated by an
increase in temperature, but the
magnitude of that effect on very
slow reactions does not seem to
have been fully appreciated. The
hydrolysis of polysaccharides, for
example,
is
shown
to
be accelerated 190,000-fold when
the temperature is raised from 25 to
100 °C, while the rate of hydrolysis
of phosphate monoester dianions
increases 10,300,000-fold.
Compounding that effect, the
slowest reactions tend to be most
heat-sensitive. These tendencies
collapse, by as many 5 orders of
magnitude, the time that would
have been required for early
chemical evolution in a warm
environment.
We propose, further, that if the catalytic effect of a
"proto-enzyme”—like that of modern enzymes—were
mainly enthalpic (panel B) rather than enthalpic (panel
A), then the resulting rate enhancement would have
increased automatically as the environment became
cooler. Several powerful nonenzymatic catalysts of very
slow biological reactions, notably pyridoxal phosphate
and the ceric ion, are shown to meet that criterion.
Taken together, these findings greatly reduce the time
that would have been required for early chemical
evolution, countering the view that not enough time has
passed for life to have evolved to its present level of
complexity.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P132
Is Leu403 an Absolute in Maintaining the “V” Conformation of Thiamin
Diphosphate in Benzoylformate Decarboxylase?
Forest H. Andrews and Michael J. McLeish
Department of Chemistry and Chemical Biology, Indiana University-Purdue University
Indianapolis, 402 North Blackford Street, Indianapolis, IN 46202-3274
Benzoylformate decarboxylase, isolated from Pseudomonas putida (PpBFDC), catalyzes the
nonoxidative decarboxylation of benzoylformate producing benzaldehyde. PpBFDC is a
member of the thiamin diphosphate (ThDP)-dependent enzyme family and, like all members,
requires ThDP to adopt a “V” shaped conformation for activity. This “V” shaped
conformation is energetically unfavorable; however PpBFDC is able to maintain ThDP in this
unfavorable conformation with the assistance of a leucine at the 403 position. Other ThDPdependent enzymes utilize hydrophobic residues such as methionine and isoleucine residue to
achieve this conformation. In an attempt to understand the requirements of the position and in
order to explore whether other residues could be used for maintaining “V” shape
conformation of ThDP, a library was constructed using site-saturation mutagenesis at Leu403.
Cell free extracts of mutants were screened for activity. Those mutants that exhibited
significant activity were purified, and kinetically characterized under steady state conditions.
Here we report the initial results from these studies.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P133
Structure and Mechanism of Benzaldehyde Dehydrogenase: A Class 3
Aldehyde Dehydrogenase from Pseudomonas putida
Michael J McLeish,1 Shreenath Prasad,1 Megan P.D. Zahniser,2 Gregory Petsko2 and Dagmar
Ringe2
1
Department of Chemistry and Chemical Biology, Indiana University Purdue University, 402
N. Blackford Street, Indianapolis, IN 46202, and 2Department of Biochemistry, Brandeis
University, 415 South St., Waltham, MA 02454
Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) is a class 3 aldehyde
dehydrogenase (ALDH).1 The class 3 ALDHs are unusual in that they are dimeric (rather than
tetrameric), relatively non-specific and unique in that they utilize both NAD+ and NADP+. In
addition, the class 3 family has been proposed to have a mechanism which differs from the
widely accepted mechanism used by other ALDH superfamily members. This alternate
mechanism was originally inspired by the rat class 3 ALDH structure,2 in which the cofactor
was found in to be in a different binding mode than that seen in any other ALDH structure
reported subsequently.
Here we present two crystal structures of PpBADH, the first structures of a class 3 ALDH
with NADP+ bound. One structure contains two dimers in the asymmetric unit and has
significant NADP+ occupancy in one monomer of each dimer. The second structure,
crystallized in the presence of the benzoate product in addition to NADP+, also contains two
dimers in the asymmetric unit, and every monomer contains NADP+ and a benzoate adduct on
the catalytic cysteine. The structures show the cofactor to be bound in the “normal” mode,
suggesting that the alternate mechanism does not apply to (at least) bacterial class 3 ALDHs.
The structures, as well as multiple sequence alignments, suggested that one of two conserved
glutamates, at the positions 215 and 337, would be important in hydride transfer. Site-specific
mutagenesis indicates that Glu215 is the more likely candidate. Although PpBADH has a
preference for NAD+ it readily utilizes NADP+. Based on our structures, sequence alignments,
and structural analysis of other NAD+- and NADP+-bound ALDHs, it appears that relatively
few amino acids are important in binding region of 2'-phosphate of NADP+. In an attempt to
rationalize the co-factor selectivity of PpBADH we have carried out site-directed mutagenesis
of several of those residues
References:
1. Yeung, C. K., Kenyon, G. L., and McLeish, M. J. (2008) Physical, kinetic and
spectrophotometric studies of a NAD(P)+-dependent benzaldehyde dehydrogenase
from Pseudomonas putida ATCC 12633, Biochimica et Biophysica Acta - Proteins
and Proteomics 1770, 1248-1255.
2. Liu, Z.J., Sun, Y.J., Rose, J., Chung, Y.J., Hsiao, C.D., Chang, W.R., Kuo, I.,
Perozich, J., Lindahl, R., Hempel, J. and Wang, B.C. (1997) The first structure of an
aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann
fold, Nat. Struct. Biol. 4 317-326.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P134
Enzymatic Extender Unit Generation for In Vitro Polyketide Synthase
Reactions: Structural and Functional Showcasing
of Streptomyces coelicolor MatB
Amanda Jane Hughes & Adrian Keatinge-Clay
Department of Chemistry and Biochemistry
University of Texas at Austin, Austin, TX 78758, USA
Correspondence: [email protected]
In vitro experiments with modular polyketide synthases (PKSs) are often limited by the
availability of polyketide extender units. To determine the polyketide extender units that can
be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional
studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this
adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender
units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS
studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and
the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45 Å and 1.43 Å
resolution, respectively. MatB crystallized in the thioester-forming conformation, making
extensive interactions with the bound extender unit products. This first structural
characterization of an adenylate-forming enzyme that activates diacids reveals the molecular
details for how malonate and its derivatives are accepted. The orientation of the α-methyl
group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize αsubstituted extender units formed by MatB before they can be accepted by PKS
acyltransferase domains. We demonstrate the in vitro incorporation of methylmalonyl groups
ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the
terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for
quantitatively monitoring certain in vitro PKS reactions using MatB is presented.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P135
Diketide Substrates Probe the Biocatalytic Potential
of Modular Polyketide Synthase Ketoreductases
Shawn K. Piasecki, Clint A. Taylor, June Liu, Jianting Zheng, Joshua F. Detelich, Arkady
Komsoukaniants, Dionicio R. Siegel, and Adrian T. Keatinge-Clay
The University of Texas at Austin, 2500 Speedway Austin, TX 78712
Modular type I polyketide synthases (PKSs) are megaenzyme complexes that are capable of
synthesizing structurally complex metabolites in an assembly line-like fashion. These
metabolites have a wide variety of uses such as antimicrobials, antifungals, insecticides and
anticancer agents[1]. The diverse set of functions makes these enzymes a useful target in the
design of combinatorial polyketide libraries. Arguably some of the most important domains
in the PKS are the ketoreductase (KR) domains, as they control the stereocenters and
therefore complexity of the growing polyketide. Isolated PKS KRs were incubated in
biocatalytic reactions with a panel of diketide substrates. The KRs include 6 A1-type KRs, 2
A2-type KRs, 1 B1-type KR, and 2 B2-type KRs, while the panel of diketides include (2RS)methyl-3-oxopentanoate N-acetylcysteamine thioester and four other diketide substrates each
one methylene group shorter or longer at the α- or γ- position. The reactions were incubated
overnight, fueled by glucose in an NADPH-regeneration system. Reduced diketide products
were analyzed by chiral chromatography. Stereochemistries of most diketide products could
be predicted from the type of KR employed. KRs that operate on smaller polyketide
intermediates within their native PKSs were observed to be more active. A2-type and B2-type
KRs maintained stereocontrol more than A1-type or B1-type KRs. Enhanced stereocontrol
was observed when N-acetylcysteamine was replaced by the more natural D-pantetheine.
These results not only improve our understanding of how stereocontrol is achieved within
PKSs, but also provide examples of how KRs can be employed as biocatalysts in the
production of chiral building blocks.
References
1. Staunton, J. and K.J. Weissman, Polyketide biosynthesis: a millennium review. Natural
Product Reports, 2001. 18(4): p. 380-416.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P136
Guilt by association: functional annotation of TM0486 from Thermotoga
maritime by identification of its bound ligands
Mark J. Snider, Brad A. Palanski and Zachary Rotter
Department of Chemistry, The College of Wooster, Wooster, OH
The structure of the TM0486 gene product from Thermotoga maritima, solved previously
without functional annotation, included extra electron density that suggested the presence of
ligands that could not be unambiguously identified. In an effort to identify the bound
ligands, as a means for establishing the function of the protein, we comprehensively screened
for ligands released from the purified recombinant protein expressed in E. coli by electrospray
ionization time-of-flight mass spectrometry. We show that thiamin, in accord with recent
findings [1], and oxidized forms of thiamin (oxythiamin and desthiothiamin) are present.
Comparative binding studies by isothermal titration calorimetry show that the hydroxymethyl
pyrimidine moiety of thiamin is important, but not sufficient, for binding. These data further
support the idea that TM0486 plays a functional role during oxidizing cellular conditions in a
novel thiamin salvage pathway. The genomic context of TM0486 includes a gene (TM0484)
coding a hypothesized ABC transporter. Current efforts are underway to establish the
substrate preference of the ABC transporter and its functional coupling with TM0486.
References:
[1] Dermoun, Z. et al. (2010) TM0486 from the Hyperthermophilic Anaerobe Thermotoga
maritima is a Thiamin-binding Protein Involved in Response of the Cell to Oxidative
Conditions. J. Mol. Biol. 400, 463-476.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P137
Aerobic catabolism of nicotinic acid in Bordetella bronchiseptica:
mechanistic studies of NicC, NicX and NicF
Mark J. Snider1, Eric Sullivan1, Matthew Henke1, Virginia Kincaid1,
Ryan Story1 and Roger Rowlett2.
1
Department of Chemistry, The College of Wooster, Wooster, OH
2
Department of Chemistry, Colgate University, Hamilton, NY
Although the biochemistry of bacterial nicotinic acid catabolism is considered a model system
for degradation of N-heterocyclic aromatic molecules, the genes coding the enzymes of this
pathway in bacteria have only recently been discerned [1]. Nicotinic acid is an intermediate
in the metabolism of coenzymes NAD/NADP and nicotine. The six enzyme-catalyzed
transformations of nicotinic acid to fumaric acid for entry into the citric acid cycle include: (1)
hydroxylation of NA to 6-HNA by NicAB, (2) oxidative decarboxylation to 2,5-DHP by
NicC, (3) oxygen insertion and ring-opening to N-FM by NicX, (4) deformylation to
maleamic acid by NicD, (5) hydrolytic deamination to maleic acid by NicF, and (6)
isomerization to fumaric acid by NicE. Using a hypothesized nic gene cluster from
Bordetella, we have cloned the nicC, nicX and nicF genes for mechanistic and structural
studies. Each His6-tagged protein has been expressed and purified from E. coli. Here we
report results of initial mechanistic studies of each enzyme, as well as the 2.4 Å crystal
structure of NicF from Bordetella bronchiseptica.
References
1. Jimenez, J. et al. (2008) Deciphering the genetic determinants for aerobic nicotinic acid
degradation: The nic cluster from Pseudomonas putida KT2440. Proc. Natl. Acad. Sci. USA
105, 11329-11334.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P138
Challenging a Paradigm. Catalysis and Inhibition in Cysteine Proteases
Michael Shokhen, Netaly Khazanov, and Amnon Albeck
Department of Chemistry, Bar Ilan University, Ramat Gan, Israel
A central mechanistic paradigm of cysteine proteases is that the His–Cys catalytic diad forms
an ion-pair NH(+)/S(-) already in the catalytically active free enzyme. The pre-activated Cys
thiolate functions as a nucleophile while the His imidazolium functions as a general acid
catalyst.
We show, by applying a computational approach, that the His–Cys catalytic diad in free
papain is fully protonated, NH(+)/SH, and therefore the experimental pKa previously
interpreted as belonging to the NH(+)/S(-)
N/S(-) equilibrium, is now assigned to the
NH(+)/SH
N/SH equilibrium.
We further simulated by molecular modeling various possible mechanisms for the reversible
formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde
inhibitors. Only one mechanism correlates with the experimental kinetic data. The His—Cys
catalytic diad is in an N/SH protonation form in the non-covalent papain-aldehyde Michaelislike complex EI. His159 is a general base catalyst, abstracting a proton from the Cys25 SH
while the activated thiolate attacks the inhibitor’s carbonyl group. The theoretically calculated
pKa’s of the catalytic His quantitatively reproduced the experimental values on the acidic and
the basic limbs of the k2/Ki vs. pH curve, and the theoretical activation barrier is very close to
a value recalculated from the experimental k-2 rate constant of the back reaction TC → EI. An
interpretation of the experimentally observed slow binding effect for peptidyl aldehyde
inhibitors is presented.
References
1. Challenging a Paradigm: Theoretical Calculations of the Protonation State of the Cys25His159 Catalytic Diad in Free Papain. Shokhen, M.; Khazanov, N.; Albeck, A. Proteins
2009, 77, 916-926.
2. The Mechanism of Papain Inhibition by Peptidyl Aldehydes. Shokhen, M.; Khazanov,
N.; Albeck, A. Proteins 2010, In press.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P139
Necrotic cell death: Is it manageable?
Keren Schultz,1 Boris Khalfin,2 Ilana Nathan,2 Amnon Albeck1
1
2
Department of Chemistry, Bar Ilan University, Ramat Gan, Israel
Department of Clinical Biochemistry, Ben Gurion University, Beer Sheba, Israel
Necrosis is one of the most common pathological processes. In contrast to apoptosis, it has
long been considered as an uncontrolled mode of cell death. Consequently, currently applied
clinical approaches are mainly focused on preventing the causes of necrosis and are very
limited once the necrotic process is initiated. Recent data on the involvement of serine
proteases in necrotic death is accumulating.
We demonstrated induction of proteolytic activity in response to treatment of two cell lines by
KCN, a known necrosis inducer, partially purified and characterized this enzyme, and studied
the effect of its inhibition on the necrotic process both in-vitro and in-vivo.
These studies may lead to a better understanding of the mechanisms of controlling necrotic
cell death, and to the development of novel anti-necrotic drugs.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P140
Computational Investigation of the Cisoid Cyclization Pathway of
5-epi-aristolocholene synthase
Carlos A. Ramirez-Mondragon and Troy Wymore
National Resource for Biomedical Computing, Pittsburgh Supercomputing Center,
300 S. Craig Street, Pittsburgh, PA 15213
The 5-epi-aristolocholene synthase from Nicotiana tobaccum (TEAS) in nature generates a
broad spectrum of sesquiterpene products via the enzyme mediated ionization of (2-trans, 6trans)-farnesyl diphosphate (FPP). The enzyme also generates trace products whose
structure/stereochemistry originates from the cyclization of (2-cis, 6-trans)-farnesyl
diphosphate(1).
Due to the inherent reactivity of the farnesyl carbocation, the precise role of the enzyme in
chaperoning the substrate and intermediates through specific pathways or alternatively what
facilitates promiscuity is difficult to ascertain. Therefore, in order to better understand
precisely how the enzyme chaperones the substrate through these alternative cisoid pathways
and determine if there are any commonalities with the transoid pathway, we have employed
both classical force field molecular dynamics simulations and hybrid Quantum
Chemical/Molecular Mechanical (QC/MM) simulations of TEAS with (cis,trans)-FPP (pdb
entry 3M02). We will present the results of computational investigation for each step of the
proposed cisoid cyclization pathway from initial ionization through formation of the (7R)-βbisabolyl carbocation, a key intermediate in the determination of the final products.
We currently propose that cisoid product formation in TEAS is both an enzyme mediated and
substrate driven process that occurs through carbocation formation and rearrangement. The
enzyme not only plays a structural role by positioning the substrate in a favorable
conformation for continued catalysis, but also actively deprotonates specific intermediates to
generate an array of minor cisoid products.
References
1. Noel, Joseph P.; Dellas, Nikki; Faraldos, Juan A.; Zhao, Marylin; Hess, B. Andes, Jr.;
Smentek, Lidia; Coates, Robert M.; O'Maille, Paul E. Structural Elucidation of Cisoid and
Transoid Cyclization Pathways of a Sesquiterpene Synthase Using 2-Fluorofarnesyl
Diphosphates. ACS Chemical Biology (2010), 5(4), 377-392
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P141
Computational Study of Glutamate-γ-Semialdehyde Oxidation Catalyzed
by Thermus Thermophilus Gamma Glutamyl Semialdehyde Dehydrogenase
Nikolay Simakov and Troy Wymore
National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center,
300 S. Craig Street, Pittsburgh, PA 15213
Gamma glutamyl semialdehyde dehydrogenase (GGSALDH, Δ1-pyrroline-5-carboxylate
dehydrogenase, ALDH4) catalyzes the oxidation reaction of glutamate-γ-semialdehyde (nonenzymatic product of Δ1-pyrroline-5-carboxylate hydrolysis) to glutamate. This reaction is
energetically coupled with reduction of NAD+ to NADH. GGSALDH is part of the proline
salvage pathway and its malfunction can lead to hyperprolinemia. One of such malfunctions
in humans is caused by a natural Ser352Lys mutation which results in type II hyperprolinemia
(mental retardation). This mutation occurs in relatively distant region from the active site and
the mechanism of how this mutation affects the enzyme function is not clear.
Here we report our current the study of the glutamate-γ-semialdehyde oxidation catalyzed by
thermus thermophilus GGSALDH and the impact of the Ser352Leu model mutation. The
human Ser352Leu mutation is modeled by respective mutation in thermus thermophilus
GGSALDH. Dynamics of the substrate and coenzyme in both the wild type and the mutant
form are studied on microsecond time scale using Anton, a specialized machine for classical
molecular dynamics (MD) simulations. Further, the mechanism of chemical reaction is
studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) approach as
implemented in pDynamo program. By combining both hybrid QM/MM calculations and the
microsecond molecular dynamic simulations, we expect to elucidate the effect Ser352Leu
mutation on protein structure, substrate and coenzyme dynamics and consequently on the
mechanism of catalyzed reaction.
References
1. Hempel J, Kraut A and Wymore T. Gamma glutamyl semialdehyde dehydrogenase:
Simulations on native and mutant forms support the importance of outer shell lysines.
Chemico-Biological Interactions, 2009, Volume 178, Issues 1-3, Pages 75-78.
2. Inagaki E, Ohshima N, Takahashi H, Kuroishi C, Yokoyama S, Tahirov TH. Crystal
structure of Thermus thermophilus Δ1-pyrroline-5-carboxylate dehydrogenase. J Mol
Biol. 2006 Sep 22;362(3):490-501
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P142
Structural investigation of a novel NRPS cluster from Pseudomonas
aeruginosa
Carter A. Mitchell and Andrew M. Gulick
Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203
University at Buffalo –SUNY
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen known for its
multidrug resistance and metabolic diversity. P. aeruginosa is a leading cause of nosocomial
infection and the number one cause of mortality in patients with cystic fibrosis. The
bacterium is capable of producing a wide range of metabolites important in quorum sensing,
virulence factors, and siderophores that scavenge key metals. Some of these metabolites are
synthesized by NRPSs.
The non-ribosomal peptide synthetases (NRPS) synthesize interesting natural products
from substrate amino acids through a high-energy adenylate intermediate followed by transfer
of the substituent to a CoA derived phosphopantetheine cofactor that is covalently bound to a
carrier protein domain (1). NRPS modules contain adenylation, peptidyl-carrier protein,
condensation, and thioesterase domains to carry out the assembly line construction of these
secondary metabolites. The P. aeruginosa genome encodes multiple NRPS synthetic clusters,
including the machinery necessary for the production of the siderophores pyochelin (2) and
pyoverdine (3), as well as the newly identified 2-amino-4-methoxy-trans-3-butenoic acids (4).
Pseudomonas aeruginosa additionally encodes several uncharacterized NRPS clusters
whose products are unknown. The operon encoded by PA1221-PA1211 is upregulated in a
Las/Rhl dependent manner and encodes a tandem adenylation-PCP domain and free-standing
enzymes with homology to NRPS condensation and thioesterase domains (5). The cluster
additionally encodes a putative acyl-CoA synthetase, a small molecule transporter, and other
tailoring enzymes. We are initiating an effort to understand the enzymes of this biosynthetic
pathway. Here we report preliminary structural and functional findings in our initial
characterization of the PA1221 operon.
References
1. Nolan EM and Walsh CT. Chembiochem. 2009 Jan 5;10(1):34-53.
2. Quadri LE, Keating TA, Patel HM, and Walsh CT. Biochemistry.1999 Nov
9;38(45):14941-54.
3. Visca P, Imperi F, and Lamont IL. Trends Microbiol. 2007 Jan;15(1):22-30.
4. Lee X, Fox A, Sufrin J, Henry H, Majcherczyk P, Haas D, Reimmann C. J Bacteriol.
2010 Aug;192(16):4251-5.
5. Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH. J Bacteriol. 2003
Apr;185(7):2080-95.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P143
Computational Exploration of Reaction Mechanism: Energetics Of DDPeptidase Using A Hybrid QM/MM With The Off-Path Simulation
Justin K. White, Jacquline Hargis, and H. Lee Woodcock
University of South Florida, Department of Chemistry, Tampa, FL 33617, USA
There is an ever growing dilemma of bacterial resistance to antibiotics; a large part of
which revolves around β-lactam based molecules. The target for this class of antibiotics is the
family of Penciling-Binding Proteins (PBPs), which are also referred to as D-alanyl-D-alanine
carboxypeptidase/transpeptidase (DD-Peptidases). Inhibiting the activity of DD-peptidases
prevents cross linking during bacterial cell wall formation. The preferential interactions
between antibiotics and the natural substrates is the focus of our investigation. The overall
mechanism and specificity of the peptidases is not entirely understood. Tipper and Strominger
suggested that a higher specificity for a β-lactams will be achieved by adding a side chain that
mimics the natural substrate1. Silvaggi, et al. designed and tested a mimetic penicillin and
cephalosporin which confirmed the previous hypothesis2. Our work explores the enzymesubstrate energetics of DD-peptidases using hybrid QM/MM techniques. Additionally, we
detail an extension of the Replica Path (RPATH) method3, Off-Path Simulation (OPS)
method, to determine reaction energetics (enthalpies and free energies) by combining reaction
path techniques with umbrella-like potentials.
References:
1) Strominger, J. L. & Tipper, D. J. (1965). Bacterial cell wall synthesis and structure in
relation to the mechanism of action of penicillins and other antibacterial agents. Am. J.
Med. 39, 708–721.
2) Silvaggi, N. R., Josephine, H. R., Kuzin, A. P., Nagarajan, R., Pratt, R. F., & Kelly, J. A.
(2005). Crystal Structures of Complexes between the R61 DD-peptidase and Peptidoglycan
-mimetic β-Lactams: A Non-covalent Complex with a “Perfect Penicilin”. J. Mol. Biol.
345, 521-533.
3) Woodcock, H. L., Hodoscek, M., Sherwood, P., Lee, Y. S., Schaefer, H. F., & Brooks, B.
R. (2003). Exploring the quantum mechanical/molecular mechanical replica path method:
a pathway optimization of the chorismate to prephenate Claisen rearrangement catalyzed
by chorismate mutase. Theor. Chem. Acc. 109, 140-148.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P144
The role of Phe161 in the mechanism of NADH-dependent persulfide
reductase from Shewanella loihica PV-4
Lee, Kyu Hyun, and Crane, EJ
Department of Chemistry, Pomona College, Claremont, CA 91711
Shewanella loihica PV-4, a psychrophilic bacteria isolated from a hydrothermal vent of Loihi
Seamount in the Pacific Ocean, can respire sulfur in a wide range of temperatures (0-42oC)
[1]. Previously in this laboratory [2] we have characterized a NADH-dependent persulfide
reductase (Npsr), which demonstrate a new reaction used in sulfur-based respiration:
R-S-S-H + NADH + H+ → H2S + R-S-H + NAD+
The steric bulk of Phe161 from the NADH-binding domain of Npsr is absolutely conserved
among Npsr and its closest homologues as Phe, Tyr, or Leu. A crystal structure of the
homologous enteroccocal NADH peroxidase suggests that this residue undergoes a significant
conformational change upon NADH binding [3]. To better characterize the reductive half
reaction of Npsr (NADH + E → NAD+ + EH2) and the mechanistic role of Phe161, we have
generated a Npsr F161A mutant by site-directed mutagenesis. A crystal structure of Npsr
F161A was determined to 2.7 Å and a NADH molecule was modeled in to correlate the
removal of steric bulk with the entropic gain of the bound NADH. Steady state kinetic
parameters of Npsr F161A were obtained and reductive titrations with various reducing
agents were carried out. Primary kinetic isotope effect (PKIE) pre-steady state kinetic
experiments with deuterated NADH (NADD) were also run anaerobically on a stopped-flow
instrument to further gain insight into the mechanistic details. The results show that the
removal of bulk at Phe161 significantly slows down catalysis, with a PKIE of 4-5. The ratedetermining step of the enzyme-catalyzed reaction also seems to be in the reductive half
reaction in the mutant unlike the wild-type enzyme. Finally, the pre-steady state data fit to
kinetic models show that the reduction of Npsr is a complicated process with at least three
steps in the mechanism. Whether upon binding NADH is entropically stabilized [5] or
enthalpically constrained to “near attack conformation” [6], it seems clear that Phe161 plays a
key role in the mechanism of Npsr.
References
[1] Gao, H. Shewanella loihica sp. Nov., isolated from iron-rich microbial mats in the Pacific
Ocean. Int. J. System Evol. Microbiology, 2006. 56: 1911-1916.
[2] Warner, M.D., Lukose, V., Lee, K., Lopez, K., Sazinsky, M., Crane, E.J. III.
Characterization of an NADH-dependent persulfide reductase from Shewanella loihica PV-4:
Implications for the mechanism of sulfur respiration via FAD-dependent enzymes.
Biochemistry, 2010. Accepted.
[3] Stehle, T., Claiborne, A., and Schulz., G.E. Eur. J. Biochem., 1993, 211: 221-226.
[4] Wallen, J.R., Mallett, T.C., Boles, W., Parsonage, D., Furdui, C.M., Karplus, P.A.,
Claiborne, A. Biochemistry, 2009, 48(40): 9650-67.
[5] Page, M.I., and Jencks, W.P. PNAS, 1971, 64(6): 1678-1683.
[6] Bruice, T.C., Lightstone, F.L. Acc. Chem. Res. 1999, 32, 127.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P145
On the accuracy of NMR-measured protein pKa values
Helen Webb, Barbara Mary Tynan-Connolly, Gregory M. Lee, Damien Farrell, Fergal
O'Meara, Chresten R. Søndergaard Kaare Teilum, Chandralal Hewage, Lawrence P.
McIntosh, Jens Erik Nielsen
School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology,
UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
Measurements of protein pKa values by NMR pH-titrations remain the experimental data of
choice for elucidating the effects of pH on enzyme active site protonation states and for
benchmarking theoretical methods for studying electrostatic effects in proteins in general.
However, the conclusions that can be derived from NMR-measured protein pKa values are
dependent on the accuracyof the experimentally measured pKa values. We re-measured the
pKa values of all carboxylic acids and His15 in Hen Egg White Lysozyme using a variety of
NMR spectra to arrive at a measure of the true experimental accuracy of NMR-measured
protein pKa values. This analysis gives insights into the true accuracy associated with
experimentally measured pKa values. We find that apparent pKa values frequently differ by
0.5 – 1.0 units depending upon the nuclei monitored, and that larger differences occasionally
can be observed in select cases. The variation in measured pKa values, which reflects the
difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization
equilibria, has significant implications for the experimental procedures used for measuring
protein pKa values, for the benchmarking of protein pKa calculation algorithms, and for the
understanding of protein electrostatics in general.
References
1. Re-measuring HEWL pKa values by NMR spectroscopy: methods, analysis, accuracy
and implications for theoretical pKa calculations
H. Webb, B. M. Tynan-Connolly, G. M. Lee, D. Farrell, F. O’Meara, C. R.
Søndergaard, K. Teilum, C. Hewage, L. P. McIntosh, J. E. Nielsen. Proteins (in
press)
2. Capturing, sharing and analysing biophysical data from protein engineering and
protein characterisation studies
Farrell D., O'Meara F., Johnston M., Bradley J., Søndergaard C.R., Georgi N., Webb
H., Tynan-Connolly B.M., Bjarnadottir U., Carstensen T., Nielsen, J. E.
Nucleic Acids Research 2010; doi: 10.1093/nar/gkq726
3. Titration_DB: Storage and Analysis of NMR-monitored Protein pH Titration Curves
Farrell D., Sá-Miranda E., Webb H., Georgi N., Crowley P.B., McIntosh L.P., Nielsen
J.E.
Proteins 2010 Mar;78(4):843-57
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P146
Crystal Structures of Both Unbound and ATP Analog-Bound Forms of
E. coli Anhydromuramic Acid Kinase,
An Important Enzyme in Peptidoglycan Recycling
C Leigh Allen1, Bradley M Hover2, Nathan I Nicely3 & Dewey G McCafferty1,2
1
Dept. of Chemistry, Duke Univ., Durham, NC. 2Dept. of Biochemistry, Duke Univ. Medical
Center, Durham, NC. 3Duke Human Vaccine Institute Shared X-Ray Crystallography Facility,
Durham, NC.
Peptidoglycan recycling is an important process that bacteria use to salvage and reuse cellular
breakdown products to avoid synthesizing new peptidoglycan de novo. This pathway involves
anhydromuramic acid kinase (AnmK), which is a simultaneous hydrolase/kinase that converts
anhydromuramic acid (anhMurNAc) to muramic acid-6-phosphate (MurNAc-6-P). The
structure of this enzyme was previously unknown, but has now been elucidated by x-ray
crystallography in both the unbound (open) and the substrate-bound (closed) forms. The
substrate synthesis was completed and a simplified substrate was prepared for use in kinetic
characterization. From the structural information, a putative mechanism was developed and
future studies aim to investigate the mechanistic enzymology of this novel kinase.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P147
Identifying the Protein Targets of Epidermal Growth Factor-Induced
Hydrogen Peroxide
Candice E. Paulsena and Kate S. Carrollb
a
Chemical Biology Doctoral Program, University of Michigan, Ann Arbor, MI 48109
b
Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458
Though initially deemed a toxic byproduct of aerobic existence, the reactive oxygen species
(ROS) hydrogen peroxide (H2O2) has been found to serve as an essential second messenger in
a multitude of signaling pathways. In response to diverse external stimuli including growth
factors, mammalian cells initiate the assembly of protein complexes that produce H2O2,
however, the cellular targets of signal-mediated ROS production remain largely unknown.
Herein we present data to demonstrate that epidermal growth factor (EGF) stimulation of
A431 cells results in H2O2 production and protein oxidation, as monitored using a cellpermeable chemical probe, which is selective for sulfenic acid. Moreover, it is shown that the
increase in both ROS production and protein sulfenylation are dependent upon EGFR
activation. Current work is aimed at identifying protein targets of EGF-mediated H2O2
production within the EGFR signaling pathway. This study constitutes the first in situ
demonstration that growth factor signaling stimulates changes in protein sulfenylation and
aims to identify unique protein targets of signal-mediated H2O2. From a chemical
perspective, this work highlights the utility of cell-permeable, small-molecule probes to
investigate redox-regulated signal transduction in living cells.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P148
Substrate Recognition and Catalytic Mechanism of MenB, the 1,4Dihydroxynaphthoate Synthase in the Menaquinone Biosynthesis Pathway
Huei-Jiun Li, Xiaokai Li, Nina Liu, Huaning Zhang, Peter J. Tonge
Department of Chemistry, Stony Brook University, Stony Brook, NY 11794 -3400
The redox cofactor menaquinone (vitamin K2) is an essential component of the electron
transport pathway in many bacteria. A central and intriguing step in the menaquinone
biosynthesis pathway is the intramolecular Claisen (or Dieckmann) condensation catalyzed by
1,4-dihydroxynaphthoate synthase (MenB) in which the naphthoquinone skeleton is formed
by the cyclization of o-succinylbenzoic acid (OSB). Although Claisen condensation reactions
typically require activation of both carboxyl groups, only one of the OSB carboxyl groups is
activated via formation of a thioester with CoA. MenB belongs to the crotonase superfamily
(CS), whose members catalyze a diverse series of reactions in which a common theme is the
stabilization of the CoA thioester oxyanion intermediate. However, MenB is a rare example of
a CS member that catalyzes C-C bond formation. Although a variety of structural and kinetic
approaches have previously been employed to characterize the mechanism of the MenB
reaction, X-ray crystallographic analysis of the active site, which lies at the interface between
two subunits, has been hindered due to the presence of a disordered loop of amino acids that
forms part of the active site. We have been able to overcome this barrier by using a stable
amino-CoA substrate mimic, and now have a well resolved image of the active site. The
crystal structure reveals critical residues involved in substrate recognition and catalysis,
information that advances our knowledge of how the enolate intermediate is generated and
how the ring closure reaction occurs within the confines of the active site. The structural data
also provides additional insight into the closely-related mechanism of BadI, another CS
member that catalyzes a ring opening, retro-Dieckmann reaction.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P149
Insights into the Mechanism of a Bacterial Quercetin 2,3 Dioxygenase
Sara Bowen, Matthew R. Schaab and Wilson A. Francisco
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604
The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative cleavage of the
heterocyclic ring of flavonols, including quercetin, to yield CO and the corresponding depside
(2-protocatechuoylphloroglucinol carboxylic acid), as shown below:
OH
OH
OH
HO
OH
O2
O
4
HO
2
3
C
OH
OH O
O
O
O
CO
OH O
Scheme 1. Reaction catalyzed by quercetin 2,3-dioxygenase
While fungal quercetinases contain one equivalent of Cu(II) per monomer (the only known
Cu(II)-containing dioxygenases), Bacillus subtilis quercetinase (BsQueD), when
overexpressed in E. coli, contains two equivalents of Mn(II). Structural studies have reveled
two separate metal-binding sites, and therefore, two possible active sites per monomer. To
understand the contribution of each site to BsQueD’s activity, the N-terminal and C-terminal
metal-binding sites have been mutated in an effort to disrupt metal binding at each site. In
wild-type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu).
All efforts to mutate His residues to non-ligating residues have resulted in insoluble protein or
completely inactive enzyme. A soluble mutant was produced when Glu 69 at the N-terminal
domain was replaced with a fourth His. This mutant (E69H) displayed 3000-fold lower
activity than that of wild-type enzyme. Further analysis of E69H by inductively couple
plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per
mol of enzyme. This is evidence that disrupting metal-ligation at one domain influences
metal-incorporation at the other domain.
In order to determine the role of Mn(II) in the catalytic mechanism of quercetinase from B.
subtilis, a structure-reactive study was conducted utilizing 4’-substituted kaempferol
derivatives (3,4’,5,7-tetrahydroxyflavone). The results of this kinetic analysis support a
catalytic mechanism for this enzyme that is similar to that proposed for extradiol catechol
dioxygenases.
This work was supported by NSF grants (MCB-0317126 and MCB-0745236 to W.A.F.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P150
Enzymatic Incorporation of Bioorthogonal Function into Proteins: New
Strategies for Microarray Construction
Rajesh Viswanathan1 and Alden E. Voelker2
Department of Chemistry, Case Western Reserve University, Cleveland OH 44106
Detection of disease states and biomolecular interactions that trigger human diseases require
tools that accurately report on cellular events.3 Due to the dominant involvement of proteins
in many significant biomedical processes, a chemical biologist benefits tremendously from
detecting their cellular status and interactions in vivo.4 Despite several layers of complexity,
site selectively modifying specific amino acid residues of native proteins offer multiple
opportunities for detection through imaging probes and microarrays.5 Recent literature has
shown promise in selectively labeling proteins through the use of bioorthogonal azide and
alkyne functional groups incorporated as substrate analogs.6 Enhanced kinetic profile
displayed by azide carrying FPP analogue inspired a similar strategy for enzymes shown to be
important in redox signaling. Our recent findings, summarized herein, document a new
approach toward the incorporation of bioorthogonal azide and alkyne functional groups
through the phenyl ring of chloro nitro benzoic acid (CNBA) and o-dinitro benzoic acid
(DNBA) as shown in Figure 1. These substrate-like small molecules have shown promising
enzymatic turnover under Glutathione S-transferase [Schistosoma japonicum, EC 2.5.1.18].
Detailed analyses (LC, LC-MS, UV and NMR) of this enzyme-catalyzed transformation will
be presented.8 Future findings will be directed towards probing the active site interactions
involved in binding and catalysis of these new analogs.7 Construction of small molecule as
well as well-defined protein arrays on SiO2 and Au surfaces are underway in our laboratory.9
Figure 1. Microarray construction
via bioorthogonal chemical ligation
R
R = Cl / NO2
R = Glutathione
R
NO2
NO2
S. japonicum GSTase EC 2.5.1.18
O
H2N
NH
H
N
COOH
SH
O
O
O
SH
O
O
S
OH
Enzyme catalysis monitored
by LC; LC-MS, UV and NMR analysis
O
N3
Au slide
References
1. Assistant Professor, Department of Chemistry, Case Western Reserve University.
2. Graduate Student, Department of Chemistry, Case Western Reserve University.
3. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature, 2004, 430, 873-877.
4. Voelker, A. E.; Viswanathan, R. Current Chemical Biology, manuscript in preparation.
5. Gauchet, C.; Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274-9275.
6. Labadie, G. R.; Viswanathan, R.; Poulter, C. D. J. Org. Chem. 2007, 72, 9291-9297.
7. Rufer, A. C. et. al. Acta Crystallogr. 2005, 61, 263-265.
8. Voelker, A. E.; Viswanathan, R. Unpublished results.
9. http://www.case.edu/artsci/chem/faculty/viswanathan/
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011
P151
Kinetic Studies of the Bi-Functional Enzyme α-AminoadipateSemialdehyde-Synthase from Bovine Liver
William E. Karsten and Paul F. Cook
Chemistry and Biochemistry Department, University of Oklahoma, Norman, OK
The enzyme α-aminoadipate-semialdehyde synthase (AASS) is a bi-functional enzyme that
catalyzes the first two steps in the degradation of L-lysine in plants and animals. The first
enzymatic activity of the bi-functional enzyme, saccharopine dehydrogenase (SDH), converts
L-lysine, α-ketoglutarte and NADPH to L-saccharopine and NADP. The second enzyme
activity (saccharopine reductase (SR)) converts L-saccharopine and NAD to α-aminoadipatesemialdehyde, L-glutamate, and NADH. We have isolated the enzyme from bovine liver and
determined the kinetic mechanism for both enzyme activities using initial rate and inhibition
studies. The kinetic mechanism of the reductase is steady-state ordered with NAD adding
prior to L-saccharopine and with NADH as the last product released. For the saccharopine
dehydrogenase, the substrates bind in the order of α-ketoglutarate, L-lysine and NADPH at
pH 8. We also observe induced product inhibition by L-saccharopine at pH 8. In contrast, at
lower pH (7.2), we observe activation of SDH by L-saccharopine and L-leucine. The
presence of L-leucine induces cooperativity in the binding of NADPH. We do not observe an
effect of L-leucine on the SR activity. We have not been able to demonstrate tunneling of
substrates in this system and hypothesize that AASS exists as a bi-functional enzyme for
regulatory purposes. We have cloned and expressed in E.coli the SDH and SR halves of the
mouse bi-functional enzyme to study the two enzymatic activities separately. The research
was supported by the Grayce B. Kerr Foundation.
22nd Enzyme Mechanisms Conference
January 2nd - 6th, 2011