Download demonstration of novel enoyl-acyl carrier protein

DEMONSTRATION OF NOVEL ENOYL-ACYL CARRIER PROTEIN
REDUCTASES IN VIBRIO CHOLERAE
BY
ROMELLE PRISCA MASSENGO-TIASSE
DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Microbiology
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2011
Urbana, Illinois
Doctoral Committee
Alumni Professor John E. Cronan Jr., Chair
Professor Jeffrey F. Gardner
Professor Charles G. Miller
Professor Abigail A. Salyers
ABSTRACT
One of the growing interests in bacterial fatty acid biosynthesis is due to the fact that an
increasing number of both synthetic and natural antibacterial compounds specifically
target fatty acid biosynthesis enzymes. However, the investigations this approach has
engendered have uncovered diversity in this relatively conserved pathway.
The enoyl-acyl carrier protein (ACP) reductase catalyses the last step in the elongation
cycle of fatty acids. It is not only a key enzyme in the pathway, but also the most diverse
enzyme across bacterial species and the target of many antimicrobial agents.
Despite this diversity, advanced bioinformatics tools failed to detect any of the known
enoyl-ACP reductase (ENR) isoforms in Vibrio cholerae. This dissertation describes the
discovery of two novel ENR isozymes by functional complementation of an Escherichia
coli fabI(Ts) mutant strain with a V. cholerae genomic library.
We first uncovered and named FabV, a new class of triclosan-resistant enoyl-ACP
reductases. In vivo and in vitro analysis of the purified protein showed that FabV is an
NADH-dependent ENR, and belongs to the same superfamily as FabI and FabL, two of
the three previously characterized ENRs. Unlike the Escherichia coli ENR (FabI), FabV
is intrinsically resistant to triclosan, the common household antibacterial agent and
expression of FabV renders its host resistant to triclosan. Interestingly, we found that
FabV is present in a number of important Gram-negative pathogens. Building on our
work, the FabV ENR activity was subsequently confirmed by other groups in two
important pathogens: Burkholderia mallei and Pseudomonas aeruginosa (Lu and Tonge
2010; Zhu, Lin et al. 2010).
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However, fabV was not the only ENR expressed in Vibrio cholerae. Screening of another
cosmid library with higher genome coverage identified yet another gene able to restore de
novo fatty acid biosynthesis in the ENR mutant strain. The product of this gene called
FabS is a novel NADPH-dependent triclosan-sensitive ENR. Unlike FabV, FabS does not
belong to any of the known superfamilies of proteins associated with known ENRs (short
chain dehydrogenase reductase, SDR or nitropropane dioxygenase, NPD). Interestingly,
the analysis of the conserved domains present in the FabS protein sequence links FabS to
NADH-quinone reductases. Comparing the presence of conserved domains present in all
known ENRs (Marchler-Bauer, Lu et al. 2011) suggests that the ENR activity may result
from convergent evolution involving protein domain shuffling. We conclude with
preliminary data suggesting the existence of other active ENRs in Vibrio cholerae,
uncovering an even greater diversity. The potential physiological relevance of such
diversity in this essential enzyme is also discussed.
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ACKNOWLEDGEMENTS
I obviously need to give credit to the many people who have one way or another
contributed to the completion of this dissertation. There are far more than even two pages
can contain, so please forgive me in advance.
I gratefully thank my advisor Dr. John E. Cronan for his generous support and guidance. I
particularly thank him for giving me a chance to join his laboratory. I also would like to
remember and thank Betsey Cronan for her generosity and kindness to us all.
Great thanks to the other members of my committee for their great help and genuine
interest in my work: Dr. Charles Miller, Dr. Jeffrey Gardner and Dr. Abigail Salyers for
her precious professional and personal advice. I also thank Dr. Kuzminov for his help as
a temporary member of my committee.
I am also thankful for Deb Lebaugh and Diana Tsevelekos for their precious help.
I obviously would like to thank my dear parents: Huberte and Maurice Massengo-Tiassé
and my so supportive siblings Ingrid and Paterne Massengo-Tiassé. I particularly thank
my mother for years of sacrifice and unwavering support.
I need to give special thanks to my adoptive grand-parents who passed away during my
PhD program: Klara Aebischer-Meyer (2007) and Othmar Aebischer (2010). I thank you
Mami for being such a dramatic influence in my life.
I give special thanks to my fiancé Jacob Yoder and the Yoder family for their support.
I also thank Tante Emmanuelle for her prayerful support, all my GradRosary friends (too
many to list here) and Gina Capra.
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I need to also thank all my past teachers, my Master’s degree advisor Dr. Chuck Wimpee
for sparking my interest in Microbiology. I also thank Dr. Imlay and Dr. Metcalf for
being such great teachers.
Last but not least, I thank God for creating this wonderfully fascinating world and for
giving us the curiosity, intelligence and persistence to explore it and slowly unveil its
secrets.
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TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION ....................................................................................... 1
1.1 PURPOSE OF THIS STUDY ................................................................................... 1
1.2 PRINCIPAL AIM ...................................................................................................... 2
1.3 SIGNIFICANCE OF THE STUDY ......................................................................... 3
1.3.1 The Fatty Acid Biosynthesis Pathway: A Valid Antibacterial Target ................ 4
1.3.2 Fatty Acids: Signal Molecules and Potential Antibiotics.................................... 4
1.3.3 Pathogenicity: A Function of Fatty Acid Profile
and Growth Medium Fatty Acid Content ..................................................................... 5
1.4 THE TYPE II FATTY ACID SYNTHESIS PATHWAY (FAS II) .......................... 7
1.5 ENOYL-ACYL CARRIER PROTEIN REDUCTASES .......................................... 8
1.6 DISSERTATION OUTLINE .................................................................................... 9
CHAPTER 2: DIVERSITY IN ENOYL-ACYL CARRIER PROTEIN
REDUCTASES ................................................................................................................. 10
2.1 ABSTRACT ............................................................................................................ 10
2.2 INTRODUCTION ................................................................................................... 11
2.3 ENOYL-ACP REDUCTASES ................................................................................ 12
2.4 DIVERSITY OF ENOYL-ACP REDUCTASES.................................................... 15
2.4.1 SDR Superfamily ENRs .................................................................................... 16
2.4.2 Non-SDR FAS ENRs ........................................................................................ 18
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2.5 PHYSIOLOGICAL MANIFESTATIONS OF ENR LEVELS............................... 20
2.6 REGULATION OF ENR LEVELS ........................................................................ 22
2.7 POLYKETIDE SYNTHASE ENRS ....................................................................... 23
2.8 ENR INHIBITORS.................................................................................................. 24
2.8.1 Inhibitors That Form a Covalent Bond ............................................................. 25
2.8.2 Non-Covalent ENR Inhibitors ........................................................................... 26
2.9 CONCLUSIONS ..................................................................................................... 30
2.10 TABLES AND FIGURES ..................................................................................... 32
CHAPTER 3: FABV DEFINES A NEW CLASS OF ENOYL-ACYL
CARRIER PROTEIN REDUCTASE IN VIBRIO CHOLERAE ...................................... 36
3.1 ABSTRACT ............................................................................................................ 36
3.2 INTRODUCTION ................................................................................................... 37
3.3 EXPERIMENTAL PROCEDURES........................................................................ 39
3.4 RESULTS ................................................................................................................ 45
3.5 DISCUSSION .......................................................................................................... 51
3.6 TABLES AND FIGURES ....................................................................................... 55
CHAPTER 4: A NEW ENOYL-ACYL CARRIER PROTEIN REDUCTASE
ISOZYME, FABS ............................................................................................................. 69
4.1 ABSTRACT ............................................................................................................ 69
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4.2 A NOVEL TRICLOSAN-SENSITIVE VIBRIO CHOLERAE
ENOYL-ACYL CARRIER PROTEIN REDUCTASE ................................................. 69
4.3 FIGURES................................................................................................................. 76
CHAPTER 5: CONCLUSIONS ....................................................................................... 80
5.1 SUMMARY............................................................................................................. 80
5.2 PRELIMINARY DATA AND DISCUSSION........................................................ 84
5.3 FIGURES................................................................................................................. 87
CHAPTER 6: REFERENCES .......................................................................................... 91
CURRICULUM VITAE ................................................................................................. 106
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CHAPTER 1
INTRODUCTION
1.1 PURPOSE OF THIS STUDY
Fatty acids are an integral part of the bacterial membrane. Their structure plays an
important role in the integrity of the membrane and its adaptation to its environment.
Very interestingly, each bacterium has a signature fatty acid profile not only specific to
its species (Bosley, Wallace et al. 1990; Landry 1994; Whittaker, Mossoba et al. 2003;
Xu, Voorhees et al. 2003), but also specific to its growth condition. Bacteria can adjust
their fatty acid composition depending on their growth conditions (Wier, Nyholm et al.;
Cronan 1975; Giles, Hankins et al. 2010). Such diversity strongly suggests a level of
diversity in the expression and regulation of genes involved in the fatty acid biosynthesis
pathway.
The purpose of this research study is to explore the diversity of the key enzyme in the
fatty acid biosynthesis pathway: the enoyl-acyl carrier protein reductase (ENR), in Vibrio
cholerae. The increasing documented diversity of this essential fatty acid biosynthesis
enzyme makes it an interesting case to study the evolution of enzymes in a given
metabolic pathway (Alves, Chaleil et al. 2002).
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1.2 PRINCIPAL AIM
Before we undertook this study, no Vibrio ENR had been reported. The main question
was: How do Vibrio organisms complete fatty acid biosynthesis since they seem to be
lacking the ENR: key player in the pathway? Two known and previously characterized
ENRs (FabI and FabL) are both members of the very large Short Chain Dehydrogenase
Reductase family (SDR), however this superfamily is so large and diverse that screening
every annotated member for enoyl-ACP reductase activity is not an efficient way to
tackle the problem. Moreover, not all enoyl-ACP reductases are members of the SDR
family. FabK, discovered in Streptococcus pneumoniae, is a flavoprotein with NADH
oxidase activity (Marrakchi, Dewolf et al. 2003). Since searching the database for
sequence homology, or even conserved domain homology had not been successful, we
screened for functional complementation of an ENR-deficient Escherichia coli strain.
The quest for different enoyl-ACP reductase (ENR) isoforms has some obvious
challenges due mainly to the fact that in silico methods are hardly infallible. Solely
relying on protein sequence homology or annotated protein functions can not only be
insufficient but also misleading. The previous discovery of the Streptococcus
pneumoniae ENR (fabK), was made difficult because the Open Reading Frame (ORF)
was wrongfully annotated, making it impossible to even suspect that the protein could be
involved in fatty acid biosynthesis. However because of the particular fact that, in S.
pneumoniae, genes involved in fatty acid biosynthesis are all gathered in a single cluster,
the mislabeled ORF seemed out of context, and was positively assayed for enoyl-ACP
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reductase activity and identified as fabK (Marrakchi, Zhang et al. 2002). Adding to the
challenge, is the fact that some genes annotated as potential ENR homologues when
tested lack ENR activity (Marrakchi, Dewolf et al. 2003) (Zhu, Lu et al. 2010). In silico
analysis is less laborious, but molecular genetics and biochemical manipulations r main
necessary.
1.3 SIGNIFICANCE OF THE STUDY
Fatty acid biosynthesis is essential not only because fatty acids are an integral part of
phospholipids that make up cell membranes, but fatty acids are also reported to have very
important roles in a number of crucial physiological processes in bacteria.
Besides insuring the integrity of membranes, fatty acid biosynthesis is also known to
provide the precursors for two essential cofactors: biotin (Lin, Hanson et al. 2010) and
lipoic acid (Jordan and Cronan 1997).
Better understanding fatty acid biosynthesis in bacteria is relevant for at least three other
major reasons: The fatty acid biosynthesis pathway is a valid antibacterial target; fatty
acids are precursors for bacterial signal molecules and potential antibiotics; the fatty acid
profile of some bacteria is linked to their pathogenicity.
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1.3.1 The Fatty Acid Biosynthesis Pathway: A Valid Antibacterial Target
The bacterial fatty acid biosynthesis pathway is a pertinent antibacterial target
(Balemans, Lounis et al. 2010). While mammals and fungi use complex multifunctional
proteins to synthesize fatty acids (Fatty Acid Synthase type I), most bacteria,
mitochondria and plants use separate proteins for each step of the biosynthesis pathway
(type II). In the type II fatty acid synthesis (FAS II), each acyl intermediate is channeled
between enzymes via an acyl carrier protein (ACP). Such basic differences between
mammals and bacteria make the FAS II enzymes specific antibacterial targets. Because
FAS II is essential, specifically disrupting this pathway will prove lethal to cells. Both
synthetic and natural compounds specifically target FAS II enzymes (Zhang, Lu et al.
2004). The ENR is the most diverse FAS II enzyme and more antibacterial compounds
specifically target the various ENRs than any other FAS II enzymes (Karioti, Skaltsa et
al. 2008). A more extensive literature review describes and discusses the relevance of the
diversity of enoyl-ACP reductases, in Chapter 2 of this dissertation.
1.3.2 Fatty Acids: Signal Molecules and Potential Antibiotics
The well-known quorum sensing signals, Acyl Homoserine Lactones (AHLs), as well as
the more recently discovered cross-kingdom cell-cell communication signals (the
Diffusible Signal Factor, DSF) are derived from fatty acids. DSF was first discovered in
Xanthomonas species which typically do not produce AHLs (Wang, He et al. 2004).
DSF-like activity was also detected in Pseudomonas and Mycobacterium species while
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this activity was inferred in Vibrio and Burkholderia species, from gene database
searches looking for RpfF, the enoyl-CoA hydratase gene involved in DSF production
(Wang, He et al. 2004). BDSF was in fact later identified in Burkholeria cenocepacia
(Ryan, McCarthy et al. 2009). DSF is a cis-11-methyl-2-dodecenoic acid and the
Burkholderia DSF (BDSF) is a cis-2-dodecenoic acid. Both types of signals are involved
in a number of biological functions, including virulence (Miller and Bassler 2001; Wang,
He et al. 2004; Ryan, McCarthy et al. 2009). Notably, BDSF was reported able to
interfere with a Candida albicans infection, demonstrating a cross-species
communication (Boon, Deng et al. 2008). Since microbes can gain an advantage over
their ecological competitors by producing antibiotics, likewise fatty acid-derived signal
molecules are of significant ecological and clinical relevance.
Interestingly, some fatty acids are also reported to have intrinsic antibacterial properties.
Short chain and medium chain fatty acids have been reported to alter virulence gene
expression in these known pathogens: Salmonella enterica Typhimurium and
Campylobacter jejuni (Boyen, Haesebrouck et al. 2008; Molatova, Skrivanova et al.
2010). The antimicrobial effect of some fatty acids was sufficient to recommend their
addition to chicken and pig feed to help reduce bacterial infection.
1.3.3 Pathogenicity: A Function of Fatty Acid Profile and Growth Medium Fatty
Acid Content
The membrane fatty acid composition of Streptococcus mutans was demonstrated to play
a significant role its virulence. A S. mutans strain unable to produce monounsaturated
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membrane fatty acids had reduced infectivity. In S. mutans, FabM is responsible for the
synthesis of monounsaturated fatty acids (Fozo and Quivey 2004). The S. mutans fabMmutant strain was significantly less infectious than the wild type strain when used to
directly infect animals (Fozo, Scott-Anne et al. 2007). The opportunistic pathogen Vibrio
vulnificus, responsible for severe wound infection and fatal septicemia after wound
infection, was shown to have decreased virulence when fatty acid biosynthesis genes
were mutated. Virulence was restored when the bacteria were supplemented with
unsaturated fatty acids during infection, strongly suggesting that the ability of V.
vulnificus to cause diseases is tied to its fatty acid metabolism (Brown and Gulig 2008).
Not only can the virulence of a bacterium be a function of its fatty acid profile, but in
some cases bacterial virulence has also been directly linked to the fatty acid composition
of the growth medium.
In Vibrio cholerae, unsaturated fatty acids in the medium were shown to increase motility
but also repress the expression of the cholera toxin (Chatterjee, Dutta et al. 2007).
Another link between fatty acids and virulence was more recently reported in V.cholerae.
The crystal structure of the ToxT protein, the transcriptional regulator ultimately
responsible for the expression of the cholera toxin, revealed a buried 16-carbon fatty acid.
This fatty acid-binding prevents ToxT from binding DNA, preventing the production of
toxin required for V. cholerae virulence. This observation directly links the growth
medium of V. cholerae to its virulence (Lowden, Skorupski et al. 2010). It is also worth
noting that V. cholerae is able to incorporate long polyunsaturated fatty acids into its
phospholipids from its environment (Giles, Hankins et al. 2010), as we previously helped
uncover in the V. fischeri squid-symbiont system (Wier, Nyholm et al. 2010).
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1.4 THE TYPE II FATTY ACID SYNTHESIS PATHWAY (FAS II)
Although most genes involved in the FASII pathway are conserved across bacterial
species, the bacteria-specific signature-fatty acid profiles and the fine-tuning of those
profiles in response to environmental conditions is consistent with a certain degree of
diversity in the FASII pathway (Marrakchi, Zhang et al. 2002).
FAS II has been well characterized in the E. coli model and most FAS II enzymes are
conserved among bacteria. The first step is the carboxylation of acetyl-CoA to malonylCoA, then FabD transacylase converts it to malonyl-ACP (Cronan and Waldrop 2002),
which is then condensed with acetyl-CoA, by FabH, to form a four-carbon 3-ketoacylACP (Lowe and Rhodes 1988). This fatty acyl-ACP will become a 16 to 18-carbon
saturated chain by entering a cycle of elongation, adding two carbons each time. This
elongation cycle comprises three steps: The reduction of the 3-ketoacyl-ACP by FabG
(Rawlings and Cronan 1992), in the presence of NADPH; the dehydration of the hydroxy
intermediate (catalyzed by FabA or FabZ) resulting in the formation of an enoyl-ACP
intermediate. Finally the formation of a saturated acyl-ACP by an NAD(P)H dependentreduction of the enoyl-ACP double bond, catalyzed by FabI, FabK or FabL (the three
identified FAS II enoyl-ACP reductases). FabB, or FabF in pathogens, condenses the
newly made acyl-ACP with malonyl-ACP to initiate another round of elongation cycle.
The fatty acid chain elongation is terminated as a result of a competition between the
acyltransferase (PlsB) and the length of the acyl-ACP (Cronan, Weisberg et al. 1975).
Typically 16 to 18-carbon fatty acids are transferred to a glycerol-3-phosphate (Green,
Merrill et al. 1981). A second fatty acid is transferred to the glycerol backbone by a
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second phospholipid acyltransferase, PlsC. Fatty acids are asymmetrically distributed on
the glycerol-phosphate to form glycerophospholipids). This distribution depends on the
specificity of the two acyltransferases.
1.5 ENOYL-ACYL CARRIER PROTEIN REDUCTASES
As mentioned above, the FAS II enoyl-ACP reductase catalyses the key last step in the
elongation cycle of saturated fatty acids. The E. coli enoyl-ACP reductase was first
identified and named EnvM, because of the deleterious effect of its mutation on the cell
envelope (e.g. release of periplasmic enzymes) (Egan and Russell 1973). Diazaborine (a
triclosan related drug) was later shown to inhibit fatty acid synthesis by specifically
targeting FabI (Turnowsky, Fuchs et al. 1989). The study of diazaborine-resistant E .coli
mutant strains linked the gene conferring the drug resistance to the envM locus. Bergler
and colleagues (Bergler, Wallner et al. 1994) demonstrated that purified EnvM was
capable of reducing an enoyl-ACP substrate, in an NADH-dependent manner. EnvM was
later renamed fatty acid biosynthesis I (FabI). Since FabI is so broadly present in bacteria,
it was originally considered the only enoyl-ACP reductase, however new isoforms were
subsequently found in other microorganisms.
The E.coli FabI and its homologues are all NADH dependent enoyl-ACP reductases.
They are all sensitive to relatively low levels of triclosan (Hoang and Schweizer 1999)
and display a characteristic Tyr-(Xaa)6-Lys motif (Rozwarski, Vilcheze et al. 1999).
Bacillus subtilis possesses two isoforms of the enoyl-ACP reductase, a FabI homologue
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and FabL. FabI could not be identified in Streptococcus pneumoniae, instead a triclosanresistant flavoprotein, FabK, with NADH-oxidizing activity, was identified as the enoylACP reductase (Marrakchi, Dewolf et al. 2003). No fabK mutants have been isolated,
suggesting that FabK is the only enoyl-ACP reductase present in S. pneumoniae.
1.6 DISSERTATION OUTLINE
Following the literature review of Chapter 2, this dissertation will describe the discovery
and characterization of a new class of triclosan-resistant ENR in Vibrio cholerae,
(chapter 3). Then Chapter 4 will report a new second triclosan-sensitive ENR. Until now,
FabI was the only known triclosan-sensitive ENR isoform. In Chapter 5, I will conclude
by describing the impact of our discovery and attempt to rationalize the functional
purpose of such ENR diversity, as well as briefly discuss the potential evolutionary steps
that may have led to such diversity.
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CHAPTER 2
DIVERSITY IN ENOYL-ACYL CARRIER PROTEIN REDUCTASES
LITERATURE REVIEW
Updated from article previously published
in “Cellular and Molecular Life Sciences”
Massengo-Tiassé and Cronan (2009), Volume 66, Number 9, 1507-1517
2.1 ABSTRACT
The enoyl-acyl carrier protein reductase (ENR) is the last enzyme in the fatty acid
elongation cycle. Unlike most enzymes in this essential pathway, ENR displays an
unusual diversity among organisms. The growing interest in ENRs is mainly due to the
fact that a variety of both synthetic and natural antibacterial compounds are shown to
specifically target their activity. The primary anti-tuberculosis drug, isoniazid, and the
broadly used antibacterial compound, triclosan, both target this enzyme. In this review,
we discuss the diversity of ENRs, and their inhibitors in the light of current research
progress.
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2.2 INTRODUCTION
Enoyl-acyl carrier protein (ACP) reductases (ENRs) catalyze the last step of the
elongation cycle in the synthesis of fatty acids. Fatty acid biosynthesis is essential for
survival in mammals, plants, fungi and bacteria (the archaea make isoprenoid-based
lipids). All these organisms use a similar set of reactions that utilize acetyl-CoA as
initiating substrate and malonyl-CoA as building block to extend fatty acyl chains by two
carbon atoms per cycle. The last step in this cycle is the reduction of the substrate enoylthioester to an acyl moiety (Figure 2.1). Although this reaction sequence is conserved
among diverse organisms, the organization and structure of the enzymes involved differ
markedly. The cytosol of mammalian cells contains a large multifunctional homodimeric
protein (called FAS I) which contains all of the enzymatic activities in the pathway
whereas the fungi have a different FAS I in which the needed active sites are split
between two multifunctional proteins that function as a hexameric structure (Cronan
2006-c). In contrast bacteria, plants, apicomplexan protozoa and mitochondria
synthesize fatty acids using a series of discrete monofunctional proteins that each carry
out a single reaction. This is called the type II fatty acid synthesis or FAS II pathway
(Bloch and Vance 1977). The FAS I and FAS II pathways are closely related in that the
high resolution structures of an individual FAS II protein can often be superimposed on
that of the cognate domain of the FAS I proteins (Maier, Jenni et al. 2006). Indeed, the
dimeric structure of the type II 3-ketoacyl-ACP synthases was an important clue in
deduction of the mammalian FAS I structure (Cronan 2004). Most of the FAS proteins
are of one generic design. For example, the structures (and to some degree the
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sequences) of the type II FabF 3-ketoacyl-ACP synthase have been conserved in all fatty
acid synthesis pathways and even in related pathways such as polyketide synthesis.
However, such conservation is not seen within the enoyl-ACP reductases. An
impressively wide diversity of proteins able to catalyze reduction of this double bond is
found in nature.
2.3 ENOYL-ACP REDUCTASES
Enoyl-ACP reductases catalyze the reduction of a trans-2-acyl-ACP (an enoyl-ACP) to
the fully saturated acyl-ACP species (note that trans-2-butyryl-ACP is often called
crotonyl-ACP). The reductant is either NADH or NADPH, although in one case a
reduced flavin (FMNH2) is used as an intermediate in the reduction. The pyridine
nucleotide reduction of the double bond is thought to proceed by conjugate addition of a
hydride ion from NADH or NADPH to carbon 3 of the trans-2-acyl group with the
intermediate formation of an enzyme-stabilized enolate anion on the C1 carbonyl oxygen
(Quemard, Sacchettini et al. 1995) (Figure 2.2). Collapse of the enolate via protonation
at C2 would yield the saturated product with the C2 proton being derived from the
hydroxyl group of an active site tyrosine side chain. The tyrosine proton is replenished
from solvent via a proton wire involving Lys163 and the ribose hydroxyl groups plus a
chain of water molecules (White, Zheng et al. 2005). The pyridine nucleotide cofactor
hydride ion utilized by the E. coli (Saito, Kawaguchi et al. 1980) and Mycobacterium
tuberculosis (Quemard, Sacchettini et al. 1995) enoyl-ACP reductases (FabI and InhA,
12
respectively) is the 4S hydrogen whereas the mammalian type I synthase uses the 4R
hydrogen (Kawaguchi, Yoshimura et al. 1980). The trans-2 unsaturated acyl chain is
linked to ACP via a thioester linkage that is required for enolization. ACP is a key
feature of the fatty acid synthetic pathway in that all of the intermediates are covalently
bound to this small, very acidic and extremely soluble protein (White, Zheng et al. 2005).
The carboxyl groups of the fatty acyl intermediates are in thioester linkage to the thiol of
the 4'-phosphopanthetheine (4’-PP) prosthetic group that in turn is linked to Ser-36 of
ACP through a phosphodiester bond. ACP thioesters are the substrates of the enzymes of
the pathway. The ACPs of type II systems are discrete proteins whereas those of the type
I systems are a domain of these polyfunctional ―megasynthase‖ proteins. These ACP
domains have a structure very similar to the type II ACPs (Heath, Su et al. 2000; Reed,
Schweizer et al. 2003; Ploskon, Arthur et al. 2008), (Reed, Schweizer et al.
2003).Although the physiological substrate of ENRs is the cognate trans-2-acyl-ACP,
these enzymes will often show activity with model substrates such as CoA or Nacetylcysteamine trans-2-acyl-thioesters. However, the KM values for the model
substrates are generally much higher than those of the ACP substrates and the Vmax
values are usually lower. In fatty acid synthesis ENRs not only complete the reaction
cycle but also serve to pull the reversible reactions that process carbon atom 3 of the acyl
chain to completion. In the absence of ENR activity, E. coli (Heath and Rock 1995) and
the mammalian type I FAS (Witkowski, Joshi et al. 2004) accumulate the products of the
3-ketoacyl-ACP synthase (the only irreversible reaction of the fatty acid synthesis cycle)
and 3-ketoacyl-ACP reductase steps as 3-hydroxyacyl-ACPs. This is due to the
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equilibrium of the 3-hydroxyacyl-ACP dehydratase step which greatly favors formation
of the 3-hydroxyacyl species.
A structure of 2-dodecenoyl-ACP cocrystallized with E. coli FabI has recently been
reported (Rafi, Novichenok et al. 2006). Unfortunately, the resolution of structure was
such that it yielded little direct information and much of the proposed structure was based
on model building and molecular dynamics simulations. Indeed, only parts of the ACP
backbone and none of the side chains were visible in the x-ray structure and these were
modeled in using the available butyryl-ACP structure. Moreover, neither the prosthetic
group nor the attached fatty acid was visible and hence these moieties were also modeled
into the structure. These manipulations plus the dynamic nature of ACP structure result
in only a speculative model. Given this caveat, the two proteins are reported to interact
via a very small interface composed of electrostatic interactions between three acid
residues of ACP helix 2 and three basic residues adjacent to the FabI substrate-binding
loop. Although this interface is similar in composition to that formed between ACP and
AcpS, it is much smaller and probably less stable. Although mutation of the implicated
FabI basic residues did result in increased Michaelis constants for 2-dodecenoyl-ACP,
some of these mutations had only modest effects and others did not conform to the purely
electrostatic interactions proposed in that substitution of alanine for a putatively key
lysine residue had a greater effect than substitution with a glutamate residue. An
interesting feature of the crystal structure is that only one of the two FabI monomers had
an associated ACP suggesting that only one of the two subunits of the FabI homodimer is
active at a time (Rafi, Novichenok et al. 2006).
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Within the field of fatty acid biosynthesis ENRs were rather neglected enzymes until the
Mycobacterium tuberculosis ENR, InhA, was discovered to be the target of the primary
anti-tuberculosis drug, isoniazid (Banerjee, Dubnau et al. 1994). This was soon followed
by the realization that the ENRs of E. coli and many other bacteria were targets of the
antimicrobial, triclosan, a synthetic compound very widely used in such every day
products as soap, toothpaste and plastics (Levy, Roujeinikova et al. 1999). These
findings argued that differences between mammalian FAS I ENRs and bacterial FAS II
ENRs rendered the bacterial enzymes good antimicrobial target candidates. However,
other bacteria were found to be resistant to triclosan suggesting the presence of triclosanresistant ENRs and these were soon demonstrated (Heath and Rock 2000) (Heath and
Rock 2000). These factors have led to a striking recent expansion of the ENR literature.
2.4 DIVERSITY OF ENOYL-ACP REDUCTASES
Most ENRs are members of the Short chain Dehydrogenase Reductase (SDR)
superfamily and thus are closely related to the other SDR enzyme of the fatty acid
synthesis cycle, 3-ketoacyl-ACP reductase, in both structure and mechanism (White,
Zheng et al. 2005). The many members of the SDR family show a significantly
conserved structure despite little sequence homology (15-30%) (Jornvall, Persson et al.
1995; Kallberg, Oppermann et al. 2002; Persson, Kallberg et al. 2003). The largely
conserved SDR folding pattern allows specific sequence motifs to be assigned, with those
for the coenzyme-binding and active site regions being the most definitive. Other classes
15
of ENRs are the TIM barrel flavin containing enzyme, FabK, the ENRs found in
mitochondria and the ENR domains of the mammalian and fungal megasynthases. The
mitochondrial ENR and the mammalian FAS ENR domain are members of the Medium
chain Dehydrogenase Reductase (MDR) family. Both NADH and NADPH are used as
the hydride source in the reactions of SDR-type ENRs. Some enzymes strongly favor
one coenzyme over the other whereas others function with either coenzyme. For
example, E. coli FabI is active with either NADH or NADPH (Bergler, Fuchsbichler et
al. 1996), but the two activities are not equally efficient. The NADPH-dependent activity
is weaker than the NADH-dependent activity and a single mutation at the C-terminal end
of the protein (Ser241Phe) results in loss of the NADPH activity (Bergler, Fuchsbichler
et al. 1996). FabI is reported to respond differently to diazaborine inhibition depending
on which cofactor is bound (Bergler, Fuchsbichler et al. 1996). The NADPH enzyme is
refractory to inhibition suggesting that FabI adopts different conformations depending on
the coenzyme bound (Bergler, Fuchsbichler et al. 1996). There seems to be no
discernable physiological rationale for coenzyme choice among ENRs, although all of the
known 3-hydroxyacyl-ACP reductases, the other pyridine nucleotide-requiring fatty acid
cycle enzyme, use NADH. Note that the coenzyme(s) used by a given SDR enzyme can
often be deduced from its sequence (Persson, Kallberg et al. 2003).
2.4.1 SDR Superfamily ENRs
The crystal structures of four SDR superfamily ENRs are available. The structures of E.
coli FabI (Baldock, Rafferty et al. 1996), the ENR of rape seed plastids (Rafferty, Simon
16
et al. 1995) (Slabas, Cottingham et al. 1990), and M. tuberculosis InhA (Rozwarski,
Vilcheze et al. 1999) indicate that all are homotetramers of essentially the same structure.
E. coli FabI and M. tuberculosis InhA are the most thoroughly studied of these enzymes.
Both have the highly conserved SDR motif, Tyr-X6-Lys in which the primary role of the
lysine side chain is to stabilize the binding of the cofactor through hydrogen bond
interactions with the hydroxyl groups of the nicotinamide ribose moiety whereas the
tyrosine acts as the proton donor to C2 (White, Zheng et al. 2005). Structures of FabI,
InhA and the plant plastid ENR complexed with various inhibitors have allowed more
detailed descriptions of the active sites of these enzymes that will be discussed below. In
comparison to the other enzymes, InhA has a deeper binding cleft that is postulated to
allow accommodation of the long-chain substrates of mycolic acid biosynthesis.
However, it has recently been shown that InhA can support short chain fatty acid
synthesis when expressed in yeast mitochondria (Gurvitz, Hiltunen et al. 2008).
Two other members of the SDR class of ENRs, FabL (Heath, Su et al. 2000) (Heath, Su
et al. 2000)and FabV (Massengo-Tiasse and Cronan 2008) illustrate the sequence
diversity within this enzyme family. Bacillus subtilis FabL is only 25% identical with E.
coli FabI and uses NADPH whereas the B. subtilis FabI protein uses NADH (Heath, Su et
al. 2000). Although FabL has the Tyr-X6-Lys motif in common with FabI proteins, the
coenzyme-binding site of FabI has the classical Rossman fold motif whereas the FabL
coenzyme-binding fold departs from that motif. Vibrio cholerae FabV is 60% larger than
the typical SDR family member (which are generally about 250 residues in length) and
the spacing between the FabV active site tyrosine and lysine residues is eight residues
(Tyr-X8-Lys) (Massengo-Tiasse and Cronan 2008) which is two more than FabI and
17
FabL and one more than the maximum reported in other SDR proteins (Persson, Kallberg
et al. 2003). However, like FabI, FabV has the classical Rossman fold motif (MassengoTiasse and Cronan 2008). A recent bioinformatic analysis of the SDR superfamily placed
the enzymes into five families (Persson, Kallberg et al. 2003). One of these families
(called divergent) is composed of the FabI-type enoyl-ACP reductases of bacteria and
plants (Persson, Kallberg et al. 2003). FabL and FabV do not fall cleanly into this or any
of the four other SDR families.
Burkholderia mallei and Pseudomonas aeruginosa are both reported to express active
FabV-type enoyl-ACP reductases (Lu and Tonge 2010; Zhu, Lin and al. 2010).
2.4.2 Non-SDR FAS ENRs
Although due to their presence in bacteria and plants ENRs of the SDR superfamily are
probably the dominant form in biology, other proteins catalyze this reaction. The most
extreme example is Streptococcus pneumoniae FabK which is an FMN-containing
protein, the crystal structure of which was recently reported. In this enzyme NADH is
the reductant, but acts indirectly by reducing the tightly bound flavin cofactor. The
reduced flavin then reduces the double bond. Unlike the SDR enzymes, FabK has a TIM
barrel (α8-β8) structure and is an FMN-dependent oxidoreductase of the NAD(P)Hdependent flavin oxidoreductase family. The closest relative of FabK is the ENR domain
of the Type I FAS of the fungi, Saccharomyces cerevisiae (Lomakin, Xiong et al. 2007)
and Thermomyces lanuginosus (Jenni, Leibundgut et al. 2007). Another close relative is
18
2-nitropropane dioxygenase which was the original annotation of FabK. FabK was
discovered due to its genomic location within a cluster of fatty acid synthetic genes and
the lack of an SDR-type ENR encoded in the S. pneumoniae genome (Heath and Rock
2000) (Heath and Rock 2000).
In mammalian and fungal mitochondria the ENRs are members of the MDR superfamily
which contains a GHE motif roughly 60 residues from the N-terminus plus a mid-chain
GX1–3GX1–3G pattern. Structures of both the yeast and human mitochondrial ENRs are
available (Airenne, Torkko et al. 2003; Chen, Pudas et al. 2008). The main function of
mitochondrial fatty acid synthesis is thought to be production of the octanoyl moiety of
lipoic acid (Jordan and Cronan 1997) (Brody, Oh et al. 1997; Wada, Shintani et al. 1997;
Gueguen, Macherel et al. 2000).
In the mammalian FAS megasynthase, in contrast to all of the other functional domains
of the fatty acid elongation cycle (Maier, Jenni et al. 2006), the ENR domain has a
different fold from its functional analogues of the bacterial type II FAS. Rather than the
SDR fold (FabI, FabL, FabV), the typical MDR fold (in mitochondrial ENRs) or even the
TIM barrel protein fold (FabK), the mammalian ER establishes a new MDR subfamily
that is structurally related to bacterial quinone oxidoreductases (Maier, Leibundgut et al.
2008). The mammalian FAS I ENR contains two subdomains, a nucleotide binding
Rossmann-fold and a substrate-binding portion. The NADP+ cofactor is bound between
the two subdomains (Maier, Leibundgut et al. 2008).
19
2.5 PHYSIOLOGICAL MANIFESTATIONS OF ENR LEVELS
The effects of ENR deficiency have been examined in several genetically accessible
organisms. In E. coli a fabI mutation that encodes an enzyme that loses activity at high
temperature blocks growth at high temperatures (Heath and Rock 1995). As expected the
growth-inhibited cells accumulated the four carbon 3-hydroxyacyl-ACP and trans-2-acylACP species (Heath and Rock 1995). Recently, it has been reported that overproduction
of FabI is toxic to growth of E. coli and this toxicity is offset by low doses of the FabI
inhibitor, triclosan, thereby indicating that overproduction of enzyme activity rather than
of FabI protein causes inhibition (Goh and Good 2008). Although the reason for this
toxicity has not been established, two hypotheses involving disruption of the normal
cellular fatty acid composition seem worth testing. The first hypothesis is that the extra
FabI activity pulls the elongation cycle such that 3-hydroxytetradecanoyl-ACP is
converted to tetradecanoyl-ACP and thus the supply of 3-hydroxytetradecanoyl-ACP
required for synthesis of lipid A, an essential outer membrane component, becomes
limiting. The second hypothesis depends on the fact that FabI potentially competes with
the isomerase activity of FabA, the dehydratase/isomerase responsible for introduction of
the double bond in E. coli unsaturated fatty acid synthesis. FabA dehydrates 3hydroxydecanoyl-ACP to trans-2 decenoyl-ACP and then isomerizes the double bond to
give cis-3-decenoyl-ACP, the double bond of which is perpetuated as the double bond of
the long chain fatty acids (Bloch 1971). However, the trans-2 decenoyl-ACP
intermediate is not tightly bound to FabA (Clark, DeMendoza et al. 1983) (Guerra and
Browse 1990) (Heath and Rock 1996) and upon dissociation could be reduced by the
20
excess FabI before it could be recaptured by FabA. This would result in an unsaturated
fatty acid deficiency which, if severe, would result in cell lysis (Cronan and Gelmann
1973). Although this scenario has not yet been tested, it is consistent with our finding
that FabI deficiency results in increased unsaturated fatty acid synthesis at the expense of
saturated fatty acid synthesis (Unpublished data). This presumably results from
accumulation of trans 2-decenoyl-ACP (Heath and Rock 1995) thereby making more
substrate available for the FabA-catalyzed isomerization reaction.
A more direct connection between ENR activity and unsaturated fatty acid synthesis
arises in Streptococcus pneumoniae. This bacterium uses a monofunctional isomerase for
introduction of the double bond in its unsaturated fatty acids (Marrakchi, Choi et al.
2002). This enzyme isomerizes trans-2 decenoyl-ACP to cis-3-decenoyl-ACP and thus
directly competes with FabK for substrate. Hence, the relative levels of the two proteins
must be closely controlled to allow the synthesis of unsaturated fatty acids, which are
essential for cell growth (Altabe, Lopez et al. 2007).
In B. subtilis, which has two ENRs, FabI and FabL, the gene encoding either enzyme can
be inactivated without blocking growth, but a strain lacking both enzymes could not be
constructed(Heath, Su et al. 2000) (Heath, Su et al. 2000). In the yeast, Saccharomyces
cerevisiae, a single mutation suffices to block growth on non-fermentable carbon sources
(Kastaniotis, Autio et al. 2004). Candida tropicalis, another yeast, has two mitochondrial
ENRs that seem to be a result of gene duplication. Either gene can replace the S.
cerevisiae enzyme in vivo. When given a mitochondrial targeting sequence the S.
cerevisiae enzyme can also be functionally replaced by E. coli FabI (Torkko, Koivuranta
et al. 2003) and M. tuberculosis InhA (Gurvitz, Hiltunen et al. 2008). The mammalian
21
homologue also restores mitochondrial fatty acid synthesis and respiratory function to the
S. cerevisiae mutant strain (Miinalainen, Chen et al. 2003). In the plant, Arabidopsis, a
mutation resulting in 90% loss of ENR activity gives only a 10% reduction in fatty acid
content indicating that Arabidopsis ENR is in large functional excess (Mou, He et al.
2000).
2.6 REGULATION OF ENR LEVELS
Although, there is some evidence for transcriptional regulation of ENR expression during
plant development (Poghosyan, Giannoulia et al. 2005) (Franz, Hatzopoulos et al. 1989;
de Boer 1996) (Franz, Hatzopoulos et al. 1989), the regulatory mechanisms remain
unclear. Differential transcription of the two mitochondrial ENRs of C. tropicalis has
also been reported specifically during growth on non-fermentable carbon source (Torkko,
Koivuranta et al. 2003), suggesting different specific roles for the isoforms, but again the
regulatory mechanisms have not been defined. In vitro results indicate that ENR activity
is regulated at the enzyme level. E. coli FabI is inhibited by long chain acyl-ACPs
(Heath and Rock 1996). This may be a case in which the product inhibition seen with all
enzymes has been utilized for regulatory purposes. E. coli FabI has also been reported to
be inhibited by palmitoyl-CoA in vitro (Bergler, Fuchsbichler et al. 1996). Although this
inhibition has been ascribed a physiological role, it remains to be demonstrated that the
observed inhibition is not due to the well-known detergent properties of long chain acylCoAs. Several bacterial FabIs were reported to be inhibited by long chain fatty acids in
22
vitro (Zheng, Yoo et al. 2005). However, since activity was assayed using a hydrophobic
model compound, trans-2-octenoyl N-acetylcysteamine, the observed inhibition might be
due to partition of the substrate into fatty acid micelles rather than a direct effect on the
enzymes. These studies should be repeated with an ACP substrate.
2.7 POLYKETIDE SYNTHASE ENRS
There is no doubt that polyketide synthesis is an evolutionary descendent of fatty acid
synthesis. The fatty acid synthase (FAS I) responsible for de novo fatty acid synthesis in
the cytosol of animal cells is homologous in sequence and analogous in architecture with
the very large family of type I modular polyketide synthases (PKSs) (Smith and Sherman
2008). Both ―megasynthase‖ assembly lines use homologous domains (ACP or PCP) to
carry the growing fatty acid or polyketide via a pantetheine-linked thioester (Smith and
Sherman 2008). The common thioester chemistry, similar structures and the adaptable
architecture have resulted in the proliferation of hybrid PKS-FAS pathways found in
phylogenetically diverse bacteria (Smith and Sherman 2008). Indeed, the extremely
complex lipids of mycobacteria are made via a partnership between a FAS and a modular
PKS (Gokhale, Saxena et al. 2007) and a class of fatty acids are produced by modular
PKS systems (Metz, Roessler et al. 2001) (Wallis, Watts et al. 2002). Therefore, a
comparison of the FAS and polyketide synthesis ENRs is of interest. The ENRs of the
type I polyketide synthases are of an MDR sub-family and are closely related to that
found in the mammalian FAS I enzyme. These ENR domains have the same location
23
within the progression of domains as the ENR of the mammalian FAS megasynthase.
Inactivation of the ENR of either a type I polyketide synthase (Bumpus, Magarvey et al.
2008) or the mammalian FAS I (Witkowski, Joshi et al. 2004) results in accumulation of
the expected trans-2 unsaturated species thereby establishing the function of these
domains.
Recently several ENRs involved in polyketide synthesis have been described and two of
these have recently been shown to have ENR activity (Bumpus, Magarvey et al. 2008).
These enzymes are either monomeric proteins or bifunctional proteins containing ENR
and acyl transferase domains. These enzymes violate the straightforward scenario of type
I polyketide synthesis in which the domains of the multifunctional megasynthase contain
all of the activities required to assemble the complete carbon skeleton. These ENRs act
on intermediates tethered to an ACP domain of the megasynthase and therefore act in
trans (Bumpus, Magarvey et al. 2008). Threading on known structures suggests that
these enzymes are related to FabK, although the presence of a flavin cofactor has not yet
been reported.
2.8 ENR INHIBITORS
A striking feature of the well-studied inhibitors of SDR family ENRs (InhA and FabI) is
that inhibition requires the NAD+ product to be bound within the active site. That is, the
inhibitor forms a stable ternary complex with the protein and the oxidized cofactor.
Some inhibitors form a covalent bond with NAD+ whereas others do not. The fact that
each of these inhibitors must complete a catalytic cycle and bind the inhibitor before the
24
weakly bound NAD+ product is released accounts for the observed slow inhibition of
ENR activity. The extensive interactions made by the inhibitors, with both the NAD+
moiety and the protein in the ternary complex, readily explains the tight binding
properties of these inhibitors.
2.8.1 Inhibitors That Form a Covalent Bond
The anti-mycobacterial properties of isoniazid (INH) have been known for almost fifty
years. The drug is active only against mycobacteria and the slow growing mycobacteria
are especially sensitive (Vilcheze and Jacobs 2007). Isoniazid-resistant strains were
isolated almost immediately after clinical use of the drug was initiated and today as much
as 30% of all clinical isolates of M. tuberculosis are isoniazid-resistant. The rapid
development of isoniazid resistance is due to the fact that the compound is a pro-drug
requiring activation by the KatG catalase/peroxidase. Most isoniazid resistance is due to
inactivating mutations within the catalase/peroxidase gene, which have no effect on M.
tuberculosis virulence. Activation of isoniazid by whole cells of M. tuberculosis
containing KatG produces a variety of isonicotinoyl-NAD adducts (Nguyen, Claparols et
al. 2001). One of these adducts, the acyclic 4S isomer, is the InhA inhibitor (Vilcheze
and Jacobs 2007). Resistance to isoniazid can occur by mutation of any one of at least
three genes other than katG and inhA (Vilcheze and Jacobs 2007) . X-ray diffraction and
mass spectrometric analyses of specific InhA NAD-acylpyridine adducts show that the
compound covalently binds C4 of the cofactor nicotinamide ring. Subsequent π-stacking
with the ring of an active site Phe residue strengthens interactions with the protein. The
25
position blocked by covalent adduct formation is that involved in the hydride transfer
from NADH to enoyl-ACP in the course of the InhA reaction cycle. Attempts to make
isoniazid-NAD+ analogues have met with some success, but this work is chemically
challenging and in its early stages (Broussy, Bernardes-Genisson et al. 2005) (Lu and
Tonge 2008).
Diazaborines are a class of heterocyclic boron-containing compounds that inhibit FabI by
the formation of a covalent bond between the boron atom and the 2’-hydroxyl of the
NAD+ ribose moiety (Baldock, Rafferty et al. 1996) (Roujeinikova, Sedelnikova et al.
1999). The drug π-stacks with the nicotinamide ring of the coenzyme and also has van
der Waals interactions with the hydrophobic substrate-binding pocket. The boron atom
and its associated hydroxyl group occupy the space of the enolate in the putative substrate
complex. While both isoniazid and diazaborines form covalent adducts with the NADbound form of enoyl-ACP reductase, the point of attachment is different and the
interaction of the two drugs with the target enzyme differs. Diazaborines seem to have
been abandoned as a medically useful set of compounds, perhaps due to their undesirable
inhibition of RNA processing in eukaryotic cells (Pertschy, Zisser et al. 2004).
2.8.2 Non-Covalent ENR Inhibitors
Triclosan (TCL)
The four types of bacterial ENRs show different interactions with TCL, a trichlorinated
biphenyl ether. In 1998 McMurray and coworkers reported that TCL resistance in E. coli
26
mapped to fabI, the gene encoding enoyl-ACP reductase (McMurry, Oethinger et al.
1998). In vivo and in vitro confirmation of this finding quickly followed(Heath and Rock
2000) (Heath and Rock 2000) . Heath and co-workers unequivocally demonstrated that
TCL inhibited the enoyl-ACP reductase step of fatty acid biosynthesis and were the first
to recognize that the specific mutations resulting in TCL resistance within fabI were
analogous to the inhA mutations imparting resistance to INH in M. tuberculosis (Heath,
Yu et al. 1998). The InhA enzymes of both M. smegmatis and M. tuberculosis were
subsequently demonstrated to also be inhibited by TCL (McMurry, McDermott et al.
1999; Parikh, Xiao et al. 2000). Likewise the Arabidopsis plant ENR was highly sensitive
to inhibition by triclosan (Dayan, Ferreira et al. 2008). Kinetic and structural studies of
the interaction of TCL with four different FabI proteins have shown that TCL forms a
tightly associated ternary complex with the protein and the charged nicotinamide cofactor
(Kastaniotis, Autio et al. 2004; Vilcheze and Jacobs 2007). Triclosan is composed of two
linked chlorine-substituted aromatic rings. As seen in the diazaborine structures (although
without covalent bond formation) the phenol ring makes π-stacking interactions with the
nicotinamide ring of NAD+ and the hydroxyl group of the drug forms hydrogen bonds
with both the phenolic oxygen of Y156 and the 2-hydroxyl of the nicotinamide ribose.
Extensive van der Waals interactions with the protein also occur. It has been proposed
that triclosan binding represents a model for substrate binding (Levy, Roujeinikova et al.
1999; Roujeinikova, Levy et al. 1999; Roujeinikova, Sedelnikova et al. 1999).
Not all SDR family ENRs are sensitive to TCL. FabL is very poorly inhibited by TCL.
The inhibition is reversible and does not require NAD+. A recent report confirms that
FabV is essentially resistant to inhibition by TCL in Burkholderia mallei (Lu and Tonge
27
2010), and confirms our suspicion that triclosan forms a rapidly reversible complex with
FabV (Figure 2.3). The FabK flavoprotein ENR is not inhibited by TCL and expression
of S. pneumoniae FabK in E. coli raises the TCL concentration required to inhibit growth
by over 4 orders of magnitude (Marrakchi, Dewolf et al. 2003). Moreover, B. subtilis
FabI is less sensitive to TCL than is the E. coli enzyme (Heath, Su et al. 2000) (Heath, Su
et al. 2000). Due to the widespread resistance of pathogenic bacteria to many of our
present antibiotics, ENRs are the focus of large scale attempts to find compounds that
specifically inhibit these enzymes. The ongoing attempts to find such molecules have
generally begun with small molecule libraries based on structures that resemble TCL
(Takahata, Iida et al. 2006; Kitagawa, Kumura et al. 2007; Takahata, Iida et al. 2007; Lu
and Tonge 2008; Tipparaju, Joyasawal et al. 2008). Some of the resulting lead
compounds inhibit FabKs as well as FabIs, although in only a few cases have the
mechanisms of inhibition been defined. However, as yet, none of these compounds has
surfaced as clinically useful antimicrobials. It should be noted that the presence of two
entire classes of enoyl-ACP reductases that are insensitive to TCL inhibition, coupled
with the promiscuous use of TCL in many everyday products, suggests it is only a matter
of time until many bacteria acquire drug-resistant ENRs. Until very recently TCL was
considered a nonspecific, broad spectrum, antibacterial and antifungal biocide. This
compound is present in all manner of consumer items, including hand soaps, mouthwash,
fabrics and plastics. The ecological damage is compounded by the fact that resistance to
TCL is often concomitant with resistance to other FabI inhibitors including isoniazid, a
drug generally reserved for use as a front line anti-tuberculosis drug. Moreover,
resistance to TCL can also arise by overexpression of MarA, SoxS, and the AcrAB-
28
encoded multidrug efflux pump, as well as by mutation of single residues within the FabI
active site. Recently,TCL has been used to select for plasmid maintenance using E. coli
fabI as the selective marker for plasmid retention (Goh and Good 2008). This system
may be of some advantage, but the possibility of resistance due to chromosomal
mutations in E. coli and the existence of naturally TCL-resistant ENRs in other bacteria
would seem significant limitations to its use.
Another possible objection to TCL, that it might inhibit the mitochondrial ENR of the
host, does not seem a problem since the bacterial and mitochondrial ENRs belong to
distinct protein families (SDR and MDR, respectively) and thus would seem unlikely to
be affected by the same inhibitors. Moreover, S. cerevisiae is insensitive to TCL unless
the yeast mitochondrial ENR is replaced with E. coli FabI. In this case the yeast cells
become sensitive to TCL indicating that the compound crosses the yeast cell and
mitochondrial membranes (Torkko, Koivuranta et al. 2003). It would be of interest to
repeat the ENR replacement experiment with the mammalian mitochondrial ENR
(Miinalainen, Chen et al. 2003) and test the TCL sensitivity of this construct. On the
other hand, TCL has been reported to inhibit the ENR activity of the type I mammalian
FAS megasynthase (Liu, Wang et al. 2002). However, TCL is approved by regulatory
authorities in the EU and the USA for many applications and an association between
bacterial triclosan resistance and antibiotic susceptibility, though suggested, has not been
found in practice (Aiello, Marshall et al. 2004).
29
Other inhibitors
Specific inhibitors have been described for FabK (Kitagawa, Ozawa et al. 2007;
Kitagawa, Ozawa et al. 2007). Kinetic analysis and a crystal structure indicate that one
such compound acts by competitive inhibition of NADH binding (Saito, Yamada et al.
2008). The diversity of the bacterial enoyl-ACP reductases relative to the lack of
structural and mechanistic diversity seen in the other enzymes of the FAS II elongation
cycle argues that naturally occurring compounds exist that selectively inhibit one or
another of these enzymes. This hypothesis has recently been confirmed by the discoveries
of natural enoyl-ACP reductase inhibitors of fungal origin that specifically target FabK
(Atromentin and Leucomelone) (Zheng, Sohn et al. 2006) and FabI proteins
(Cephalochromin) (Zheng, Sohn et al. 2007) and cyperin (Dayan, Ferreira et al. 2008).
The mechanisms of ENR inhibition by these antibiotics remain to be described in detail.
It is interesting that Cephalochromin was identified over 20 years ago as a natural
compound of fungal origin with anti-tumor properties (Koyama, Ominato et al. 1988).
2.9 CONCLUSIONS
The diversity of ENRs in nature is surprising and suggests that evolutionary pressures
such as the natural inhibitors mentioned above have exerted a selection for diversity. A
sign of the evolutionary pressures is that some bacteria have only a single ENR whereas
others have two, either of which suffices for growth in the laboratory. If these ENRs are
30
functionally redundant, why are both enzymes retained? This must be understood before
ENR inhibitors with broad spectrum antibacterial activity can be developed. Moreover, it
is likely that additional protein families that carry out the ENR reaction will be
discovered. This seems certain to occur if ENR inhibitors turn out to be clinically useful
antimicrobials. Finally it should be noted that annotation of bacterial ENRs is often
problematical. Genomes generally encode many SDR family members and in the
absence of a genomic context consistent with a role in fatty acid synthesis, a true ENR
can be very difficult to distinguish from other family members that carry out the
unrelated reactions such as reduction of a keto group. A parallel problem arises with the
other SDR family fatty acid synthetic enzyme, 3-ketoacyl-ACP reductase (Wang and
Cronan 2004). It is also difficult to identify FabK homologues. Several proteins that
align well with S. pneumoniae FabK have been reported to lack ENR activity (Heath, Su
et al. 2000; Marrakchi, Dewolf et al. 2003).
31
2.10 TABLES AND FIGURES
Name
Catalytic signature
References
(Jornvall, Persson et al. 1995;
Most SDR members
Asn-Xn-Ser-X10-Tyr-X3-Lys
Filling et al. 2002)
M. tuberculosis InhA
Phe-X8-Tyr-X6-Lys
(Rozwarski et al. 1999)
S. typhymurium FabI
Ser-X10-Tyr-X6-Lys
(Rozwarski, Vilcheze et al.
1999)
H. pylori FabI
E. coli FabI
(Rozwarski, Vilcheze et al.
B. subtilus FabI
Thr-X10-Tyr-X6-Lys
1999)
B. subtilus FabL
Ser-X10-Tyr-X6-Lys
(Heath, Su et al. 2000)
(Massengo-Tiasse and Cronan
V. cholerae FabV
B. mallei FabV
Ser-X10-Tyr-X8-Lys
2008; Lu and Tonge 2010;
Zhu et al. 2010)
P. aeruginosa FabV
B. napus ENR
A. thaliana ENR
Thr-X10-Tyr-X7-Lys
(Rozwarski, Vilcheze et al.
1999)
Nicotinia tabacum ENR
Table 2.1 Comparison of the catalytic signatures of enoyl-ACP reductases members of
the large Short chain Dehydrogenase Reductase superfamily. In bold are the bacterial
ENRs and the bottom three are plant ENRs.
32
Figure 2.1 The enoyl-ACP reductase (ENR) reaction. The bond reduced is shown in red.
The lengths of the red bonds are accentuated for purposes of illustration. Depending on
the enzyme the reductant can be NADPH or FMNH2 rather than NADH.
33
Figure 2.2 The reaction mechanism of E. coli FabI. The double bond reduction occurs
by conjugate addition of a hydride ion from NADH (or NADPH) to C3 of trans-2butenoyl-ACP. The hydrogen added to C3 is the pro-S hydrogen of the coenzyme. The
Tyr156 proton is replenished by a proton relay system through Lys163 and the hydroxyls
of the coenzyme ribose moiety plus a chain of water molecules that provide access to
solvent.
34
Figure 2.3 Overview of triclosan effect on the SDR family ENRs, FabI, FabL and FabV.
NAD binds to FabI only weakly, while FabV is strongly inhibited upon preincubation of
the enzyme with NAD in the absence of substrate (Massengo-Tiassé and Cronan 2008).
Triclosan (Tcl) does not directly bind to any of the three enzymes with appreciable
affinity. The FabI-NAD-triclosan ternary complex is stable and highly inhibitory
whereas FabL is unable to form a stable ternary complex.
Although we suspected a very rapid reversible inhibition, whether triclosan was actually
able to bind the unstable FabV-NAD complex was unclear, but of little relevance since
FabV was essentially resistant to Tcl. Lu and Tonge (2010) have since demonstrated that
Burkholderia mallei FabV does form a rapid and reversible FabV-NAD-Tcl ternary
complex.
35
CHAPTER 3
FABV DEFINES A NEW CLASS OF ENOYL-ACYL CARRIER PROTEIN
REDUCTASE IN VIBRIO CHOLERAE
Modified from article previously published in the “Journal of Biological Chemistry”
Massengo-Tiassé and Cronan (2008), Volume 283 Number 3, pp. 1308–1316
3.1 ABSTRACT
Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid
elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia
coli that is the target of the antibacterial compound, triclosan. However, some Grampositive bacteria are naturally resistant to triclosan due to the presence of the triclosanresistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gramnegative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the
three known enoyl-ACP reductase isozymes suggesting that this organism encodes a
novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene
encoding the new isoform, called FabV, was isolated by complementation of a
conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid
synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a
member of the short chain dehydrogenase reductase superfamily, although it is
36
considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues).
The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires
many gaps and yields only 15% identical residues. Thus, FabV defines a new class of
enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is
active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI
and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of
FabV in E. coli results in markedly increased resistance to triclosan and the purified
enzyme is much more resistant to triclosan than is E. coli FabI.
3.2 INTRODUCTION
Fatty acid synthesis is essential for the formation of membranes and hence for the
viability of all cells excepting the Archaea. The bacterial fatty acid synthesis system
(FAS II) differs significantly from the mammalian and fungal system (FAS I). Whereas
FAS I systems use complex multifunctional proteins to synthesize fatty acids, bacteria
use separate discrete proteins for each step of the biosynthesis pathway (Cronan 2006-a;
Cronan 2006-c). In the FAS II systems of bacteria, chloroplasts, apicoplasts, and
mitochondria, each acyl intermediate is channeled among enzymes as an acyl carrier
protein (ACP) thioester. The differences between the FAS I and FAS II systems make the
FAS II enzymes good targets for antibacterial inhibitors (Campbell and Cronan 2001;
Payne, Warren et al. 2001; Heath and Rock 2004; Zhang, White et al. 2006). The
paradigm FAS II system is that of Escherichia coli and this has provided an excellent
37
model system. Most FAS II enzymes are relatively conserved among bacteria and some
domains of the FAS I proteins are clearly derived from FAS II proteins (Jackowski and
Rock 1987). An exception is the last step of the elongation cycle (Figure 3.1) formation
of a saturated acyl-ACP by an NAD(P)H-dependent reduction of the enoyl-ACP double
bond. In E. coli this reaction is catalyzed by the product of the fabI gene, which was
discovered as the target for the antibacterial action of a set of diazaborine compounds.
FabI was later shown to also be the site of action of triclosan (McMurry, Oethinger et al.
1998), an antibacterial compound used in hand soaps and a large variety of other
everyday products. Although FabI homologues are widely distributed in bacteria and
other FAS II-containing organisms, the existence of a number of bacterial species
naturally resistant to triclosan was soon recognized. In these bacteria triclosan resistance
was due to the presence of other enoyl-ACP reductase isozymes of varying resistance to
triclosan. Bacillus subtilis contains two isozymes, a FabI homologue and a somewhat
triclosan-resistant isozyme called FabL (Heath, Su et al. 2000) (Heath, Su et al. 2000)
that, like FabI, is a member of the short chain dehydrogenase reductase superfamily.
Streptococcus pneumoniae contains a single enoyl-ACP reductase, FabK, that is
refractory to triclosan and is a flavoprotein unrelated to the short chain dehydrogenase
reductase isozymes (Heath and Rock 2000).
Vibrio cholerae is a Gram-negative bacterium that causes cholera in humans. When we
began our work only a single genome sequence was available for this organism
(Heidelberg, Eisen et al. 2000) and this sequence argued that V. cholerae did not encode a
convincing homologue of any of the known enoyl-ACP reductase isozymes (FabI, FabK,
or FabL). This conclusion was recently confirmed by shotgun genome sequences of a
38
large number of V. cholerae strains now available from the NCBI genomes data base.
Moreover, the genome sequences of other Vibrio species also show the lack of known
enoyl-ACP reductase homologues. The lack of a FabI homologue was especially
surprising because otherwise the Vibrio FAS proteins are very similar to those of E. coli.
Indeed, the major FAS II gene clusters of E. coli and V. cholerae have almost identical
organizations. We report the isolation of the gene that encodes a fourth enoyl-ACP
reductase isozyme that we have named FabV and some properties of the new enzyme.
3.3 EXPERIMENTAL PROCEDURES
Construction of Bacterial Strains and Plasmids—E. coli strain JP1111, which carried the
temperature-sensitive (Ts) fabI392 allele, was obtained from the Coli Genetic Stock
Center, Yale University. The mutation, which will be called fabI(Ts), allows growth at 30
°C but blocks growth at 42 °C. The temperature-sensitive activity of the mutant enzyme
has been directly demonstrated (Bergler, Fuchsbichler et al. 1996) and is due to a single
point mutation (Bergler, Hogenauer et al. 1992). Strain EPI300 (genotype recA1 endA1
araD139 Δ(ara,leu)7697 trfA, with the trfA gene being under arabinose regulation) was
made temporarily proficient in homologous recombination by introducing plasmid
pEAK2, which carries a functional recA gene on an unstable plasmid (Kouzminova,
Rotman et al. 2004). To move the fabI(Ts) mutation from JP1111 into this strain a closely
linked Tn10 transposon insertion was introduced into the JP1111 chromosome by
transduction to tetracycline resistance at 30 °C with a phage P1vir stock grown on the
39
trpC::Tn10 strain CAG18455 (Singer, Baker et al. 1989; Nichols, Shafiq et al. 1998)
followed by screening for recombinants that remained temperature sensitive. A phage
P1vir stock grown on one of these recombinants was then used to transduce the EPI300
(pEAK2) strain to tetracycline resistance at 30 °C with screening for temperature
sensitivity and loss of the pEAK2 antibiotic resistance determinant to give strain PMT03.
For plasmid constructions the primers listed in supplemental Table 3.1S were used to
amplify the fabV gene from the complementing cosmid using Pfu Turbo® DNA
polymerase (Stratagene). The PCR products were directly cloned into the expression
vectors as given in supplemental Table 3.1S using the EcoRI and PstI restriction sites
included in the primer sequences. Cloning into pET101 was performed using the
Champion pET101 Directional TOPO expression kit (Invitrogen). Plasmids expressing
the native form of fabV were obtained by use of primers CtagVcfor and NtagVcREV for
insertion into pET101 and primers EcoVCfor and PstVCREV for insertion into
pBAD322. The plasmids expressing the C-terminal His6-tagged form of FabV were
obtained by use of primers CtagVcfor and NtagVcREV for insertion into pET101 and
primers EcoVCfor and pBADCtagVc rev for insertion into pBAD322. The pBAD322derived plasmid that expressed the N-terminal His6-tagged form of FabV was obtained
by use of primers pBADNtagVc and PstVCREV. The plasmid constructions were
verified by DNA sequencing performed by the Keck Genomics Center of this institution.
Cosmid Library Production and Subcloning—The CopyControl Fosmid Library
Production Kit from Epicentre (Madison, WI) was used to generate an unbiased V.
cholerae genomic library. The pCC1FOS vector carries two origins of replication, a
single copy origin (ori2) and an inducible high copy origin (oriV) (Wild, Hradecna et al.
40
2002). Genomic DNA from cells of the V. cholerae biotype albensis strain ATCC 14547
was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI).
The DNA fragments were obtained by mechanically shearing genomic DNA by 20
passages through a 200-μl pipette tip. The resulting DNA fragments were end-repaired to
5′-phosphorylated blunt ends and then separated on a 0.8% low melting point-agarose gel
run overnight at 30 V. Sheared DNA fragments of about 40 kb were size selected, gelpurified, ligated to the dephosphorylated pCC1FOS vector, and then packaged into λ
phage particles. This was used to transduce the fabI(Ts) E. coli recipient strain PMT03 to
chloramphenicol resistance at 30 °C under conditions where the entering plasmids would
replicate at single copy. The resulting transductants were then tested for the ability to
grow at 42 °C, the nonpermissive temperature of the fabI(Ts) allele. A complementing
clone that grew well at 42 °C was obtained. The purified complementing cosmid was
again transformed into the fabI(Ts) strain PMT03 to confirm the complementation. This
strain was then induced with arabinose to induce the cosmid to high copy number and
large quantities of the complementing cosmid DNA were obtained using the Qiagen
Large-construct Kit (Valencia, CA). The cosmid was then partially sequenced using
vector-specific primers to localize the insert DNA on the V. cholerae genome. To obtain
smaller complementing clones 50 μg of the 40-kb cosmid was sonicated for 5 seconds,
and the DNA fragments were end-repaired before ethanol precipitation in the presence of
glycogen. Fragments larger than 3 kb were gel-purified, cloned into the pCC1FOS vector.
The resulting plasmids were then transformed into mutant strain PMT03 and screened for
complementation at 42 °C and for triclosan resistance at 30 °C. Each complementing
subclone was completely sequenced and its genomic location identified by a BLAST
41
search against the V. cholerae El Tor N16961 genome (Heidelberg, Eisen et al. 2000)
using the TIGR Comprehensive Microbial Resource website (cmr.tigr.org).
Enzyme Purification—Primers were designed for cloning the candidate gene into a
pET101 expression vector from the Invitrogen Champion pET101 Directional TOPO
Expression Kit (Carlsbad, CA). Six histidine codons were added at the 3′-end of the gene
to produce a His tag at the C-terminal end of the protein. The gene was PCR-amplified
using Pfu Turbo DNA polymerase from Stratagene (La Jolla, CA). The PCR fragment
was cloned into pET101, sequenced, then transformed into BL21 Star (DE3) chemically
competent cells from Invitrogen. The expression of the cloned fragment was induced
with 150 μM isopropyl β-d-thiogalactopyranoside. The crude extract was then applied to
a nickel-nitrilotriacetic acid spin column from Qiagen (Valencia, CA), washed with the
lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole), and the reductase
eluted with 250 mM imidazole in the same buffer. Similarly, the native protein (lacking a
histidine-tag) was also cloned and overexpressed from the pET101 vector. Purification
was first performed through a Vivapure D maxi H spin column from Vivascience
(Sartorius). The protein was washed with 4 mM Tris-HCl, 1 mM EDTA (pH 8) lysis
buffer, discontinuously eluted with a 50 mM Tris-HCl (pH 7.5) buffer containing 100,
200, 300, 400, 500, 700, or 1000 mM NaCl. Most of the reductase was eluted with 400
mM NaCl. The fractions eluted at 400 and 500 mM NaCl were pooled, then desalted by
overnight dialysis before passing through a Blue Sepharose column from using the
ÅKTA purification apparatus (GE Healthcare). Elution from the affinity column was
achieved using a 2 mM LiCl buffer solution after washing with 15 column volumes of 20
mM sodium phosphate buffer (pH 7). Finally the protein was concentrated by
42
ultracentrifugation using a 30,000 MWCO Amicon Ultra centrifugal filter device from
Millipore.
Fatty Acid Synthesis Assays—The ability of the new gene to restore in vivo fatty acid
synthesis was tested by transforming the fabI(Ts) mutant strain, JP1111 with a plasmid in
which the gene encoding the putative V. cholerae enoyl-ACP reductase had been inserted
into the arabinose inducible pBAD322 vector (Cronan 2006-b). The cultures were grown
at permissive temperature, induced with arabinose, shifted to 42 °C, and then [114C]acetate (specific activity, 55 mCi/mmol) from American Radiolabeled Chemicals,
Inc. (St Louis, MO) was added as described in Lai and Cronan (Lai and Cronan 2004).
Labeled phospholipids were extracted, run on a thin layer chromatography plate, and
visualized after overnight exposure to x-ray film (Lai and Cronan 2004). The in vitro
fatty acid synthesis assay was performed using a mixture of 0.1 M sodium phosphate
buffer (pH 7), 0.1 M LiCl, 50 μM acetyl-CoA, 25 μM holo-ACP, 150 μM NADH, 1 mM
2-mercaptoethanol, 200 μg of ammonium sulfate fraction of a cell extract (Jackowski and
Rock 1987), and 5 μg of pure C-terminal (His)6-tagged FabV protein (when added).
Following addition of 25 μM [2-14C]malonyl-CoA (specific activity, 51 mCi/mmol), the
200-μl reactions were incubated at 37 °C for 45 min (Lai and Cronan 2004).
Radiolabeled malonyl-CoA was purchased from Moravia Biochemicals, Inc. (Brea, CA).
Free fatty acids were extracted by saponification followed by acidification and petroleum
ether extraction as reported by Gelmann and Cronan (Gelmann and Cronan 1972).
Cell-free Extract Preparation—Cell cultures of wild type E. coli and the fabI mutant
strain JP1111 were centrifuged and resuspended in lysis buffer (0.1 M sodium phosphate,
pH 7.5, mM 2-mercaptoethanol, 1 mM EDTA) before lysis through a French pressure
43
cell. Partial purification of fatty acid synthetic enzymes was performed by gradual protein
precipitation of each lysate with ammonium sulfate. The same procedure was used to
prepare V. cholerae extracts. In the case of strain JP1111 the cultures were shifted from
30 to 42 °C for 15 min before harvesting to inactivate the mutant FabI protein.
Preparation of Holo-ACP, Apo-ACP, and Crotonyl-ACP—Holo-ACP was purified
following the procedure described by Thomas and Cronan (Thomas and Cronan 2005).
The purification of apo-ACP was identical to that of holo-ACP, except that AcpH, rather
than AcpS, was overexpressed along with AcpP to convert the holo-ACP species to apoACP. Crotonyl-ACP was enzymatically synthesized from crotonyl-CoA purchased from
Sigma using B. subtilus Sfp phosphopantetheinyl transferase (Quadri, Weinreb et al.
1998) expressed from pNRD136. In a 30-ml reaction volume, 25 mg of crotonyl-CoA, 1
mM dithiothreitol, 30 mg of apo-ACP, and 2 mg of Sfp enzyme were mixed in a buffer
containing 50 mM Tris-HCl (pH 8.8), 10 mM MgCl2 and then incubated for 4 h at 37 °C.
The reaction mixture was then precipitated with 5% trichloroacetic acid and 0.02%
deoxycholate resuspended, dialyzed, and concentrated with an Amicon
ultracentrifugation filter device from Millipore (5,000 MWCO) (Thomas and Cronan
2005). Crotonyl-ACP synthesis was verified on a 20% native gel containing 2.5M urea
and by electrospray mass spectrometry (observed mass 8915.39; calculated mass
8915.09).
NADH Oxidation Assay—Enoyl-ACP reductase activity was monitored
spectrophotometrically by decrease in absorbance at 340 nm using an NADH extinction
coefficient of 6220 M–1. Each 100-μl reaction was performed in disposable UVtransparent microcuvettes obtained from BrandTech Scientific, Inc. The activity assays
44
contained varying concentrations of NADH, 1 pg of the purified native FabV, varying
substrate concentrations (crotonyl-CoA or crotonyl-ACP), and 0.1 M LiCl in a 0.1 M
sodium phosphate buffer (pH 7). Kinetic constants were determined using GraphPad
PRISM version 4 software. The Km values for NADH and NADPH were determined at a
crotonyl-ACP concentration of 80 μM. The Km values for crotonyl-ACP and crotonylCoA was determined using 150 μM NADH. Triclosan was purchased from KIC
Chemicals, Inc. (Armonk, NY).
Size Exclusion Chromatography—The native FabV was chromatographed on a Superdex
200 HR 10/30 column in a 0.15 M NaCl, 50 mM sodium phosphate buffer at pH 7 on the
AKTA purifier (GE Healthcare). The calibration curve was prepared using
chymotrypsinogen, ovalbumin, bovine serum albumin, aldolase, ferritin, and
thyroglobulin as standards ranging from 25,000 to 669,000 Da. The FabV peak was
detected by absorbance at 280 nm and the reductase activity in the fraction was
confirmed by spectrophotometric assay and the size of the monomer was confirmed by
8% SDS-PAGE of the peak fractions.
3.4 RESULTS
Identification of a V. cholerae gene Encoding a Novel Enoyl-ACP Reductase—A cosmid
was selected from the V. cholerae genomic library that consistently complemented
growth of the fabI(Ts) strain PMT03 at 42 °C (Figure 3.2A). The large V. cholerae
genomic fragment (about 40 kb) carried by the cosmid was fragmented and the fragments
45
were cloned into the same vector to obtain smaller complementing plasmids. Four of
these subclones were able to complement growth of the fabI(Ts) mutant strain at 42 °C
and only those clones also allowed growth on triclosan at 30 °C (Figure 3.2B). Upon
partial sequencing, all four clones were found to contain a common genomic region that
contained only three open reading frames (ORFs) by comparison with the genome
sequence of V. cholerae O1 biovar El Tor. N16961, the only sequenced V. cholerae
genome then available. One of the three ORFs (VC1737) was annotated as encoding
protein synthesis initiation factor IF-1, whereas the other two ORFs, VC1738 and
VC1739, were annotated as hypothetical proteins (Figure 3.10). The latter two ORFs had
been predicted to be in a single transcriptional unit (www.BioCyc.org). To identify these
ORFs, a BLAST search of each of the ORFs was performed against all of the bacterial
genomes in the data base. Both VC1738 and VC1739 showed homology to a single ORF
found in a number of bacterial genomes. These were (accession number follows the
species name) Xanthomonas campestris (YP361851), Vibrio parahaemolyticus
(NP797610), Vibrio vulnificus (NP761639), Vibrio fischeri (YP204271), Shewanella
oneidensis (NP717408, Shewanella denitrificans (YP_563490), Aeromonas hydrophila
(YP857138, Xylella fastidiosa (NP779243), and Photobacterium profundum (YP130609).
That is, when one of the V. cholerae N16961 ORFs aligned with an ORF of one of these
bacteria, the other V. cholerae N16961 ORF aligned with that same ORF without overlap.
Hence, the original V. cholerae genomic sequence seemed to contain a frameshift
introduced by a sequencing error that had cut a single ORF into two ORFs. We fully
sequenced the insert of one of the complementing V. cholerae subclones and found that
the VC1738 and VC1739 ORFs were indeed a single ORF consistent with a prediction by
46
the Swiss-Prot data base. The error was omission of a single base within a run of six
adenine bases on the coding strand (Figure 3.10). This was confirmed by the finding that
the genome fragment containing the putative VC1738 and VC1739 ORFs expressed only
one protein of the molecular mass (45 kDa) expected from elimination of the frameshift
(see below). Our sequencing data were subsequently confirmed upon submission of
shotgun genomic sequences of a large number of V. cholerae strains to the NCBI Whole
Genome Shotgun (wgs) data base. All show that the original VC1738 and VC1739 ORFs
lie within a single protein. The comparative analysis of the protein sequence encoded by
our candidate gene extended the presence of homologues of our candidate protein to
Xanthomonas oryzae (YP449055), Xanthomonas axonopodis (NP640497), Pseudomonas
putida (NP746744), Pseudomonas entomophila (YP610084), Yersinia pestis
(NP407525), Yersinia pseudotuberculosis (YP072425), Pseudomonas aeruginosa
(NP251640), Saccharophagus degradans (YP528097), Methylobacillus flagellatus
(YP545785), Burkholderia species (e.g. YP443187) Cytophaga hutchinsonii,
Pseudoalteromonas species (e.g. NC008228), Clostridium species (NC008261),
Idiomarina loihiensis (NZAAMX01000026), Treponema denticola (NC002967), and
Streptomyces avermitilis (NC003155). These alignments range from 96.5% protein
identity for the V. fischeri homologue, to 52.9% residue similarity for the Streptomyces
avermitilis homologue. We have named the gene fabV and the encoded protein FabV
because it is predominately found in Vibrio species and closely related organisms where
it seems to be the sole enoyl-ACP reductase. However, due to the diverse reactions
catalyzed by the enzymes of the short chain dehydrogenase reductase (SDR) superfamily
some of the more poorly aligned proteins may not be enoyl-ACP reductases. Several of
47
the above bacteria are human pathogens and others infect plants and marine organisms.
There may be bacteria that have both FabV and FabK such as Clostridium tetani.
However, like FabV various putative FabK homologues may not possess enoyl-ACP
reductase activity (Marrakchi, Dewolf et al. 2003). FabV, like FabI and FabL, is a
member of the SDR superfamily (Duax, Griffin et al. 1996; Persson, Kallberg et al. 2003)
although FabV aligns only weakly with FabI or FabL even when many gaps are allowed
(Figure 3.3). FabV does not contain the Tyr-(Xaa)3-Lys motif typical of most members of
the SDR superfamily. FabI and FabL also lack this motif and instead use a Tyr-(Xaa)6Lys active site motif. In FabV another variation is seen, a Tyr-(Xaa)8-Lys motif (Figure
3.3). Given the conservation of residues that neighbor the key tyrosine and lysine
residues this seems likely to be the FabV active site although this remains to be tested by
mutagenesis. Despite the low homology scores FabV can be modeled on the crystal
structures of known SDR enoyl-ACP reductases by the Geno3D server (geno3dpbil.ibcp.fr), which takes into account known protein structures as well as predicted
geometrical restraints (Combet, Jambon et al. 2002).
The Enzymatic Activity of FabV—Expression of FabV in a fabI(Ts) mutant strain restored
de novo fatty acid synthesis at the non-permissive temperature to wild type levels (Figure
3.4). Moreover, addition of purified FabV (see below) to cell extracts of fabI(Ts) mutant
strains gave wild type rates of in vitro fatty acid synthesis (Figure 3.11). These early in
vitro experiments were done with a version of FabV carrying a His6 purification tag and
purified by Ni2+-chelate chromatography (see below). Later we found that under
conditions of low expression (where protein production is stochastic among cells
(Rosenfeld, Young et al. 2005; Yu, Xiao et al. 2006) the survival of an E. coli fabI(Ts)
48
mutant strain expressing this construct was much lower than that of the same strain
expressing the native form of the protein (Figure 3.5). Because the N-terminal His6tagged FabV also gave inefficient complementation (albeit to a much lesser extent), we
overexpressed the native FabV in E. coli and purified it from that source by two
chromatographic steps (Figure 3.6). The most effective step in the purification of the
native protein was chromatography on Blue Sepharose CL-6B where the immobilized
Cibacron Blue F3G-A dye mimics adenylate-containing cofactors such as NAD . A
similar step was used in the purification of E. coli FabI (Bergler, Wallner et al. 1994).
The native FabV was active on both crotonyl-CoA and crotonyl-ACP using the standard
NADH oxidation spectrophotometric assay and thus commercially available crotonylCoA was often used to assay the enzyme (Figures 3.7 and 3.8). The native FabV was
much more active then the C-terminal His6-tagged form (Figure 8A) and showed a very
strong preference for NADH over NADPH when assayed with crotonyl-CoA (Figure 8B).
The Km for NADPH was 60-fold higher than that for NADH (Table 3.1). The
experimentally observed maximal velocities of FabV with crotonyl-CoA and crotonylACP were similar with the rate with the latter substrate being about 50% higher. The Km
for crotonyl-ACP (195 μM) was 6-fold lower than that for crotonyl-CoA (1178 μM)
(Table 3.1). It should be noted that the ACP used was that of E. coli rather than that of V.
cholerae. However, the ACP of V. cholerae is 96% identical to that of Vibrio harveyi,
which has been shown to functionally replace the ACP of E. coli in vivo (De Lay and
Cronan 2007) (the ACPs of V. cholerae and E. coli are 84% identical).
49
FabV is a monomeric protein in solution as determined by size exclusion chromatography
(Figure 3.6). This is in contrast to FabI, which is a homotetramer. The solution structure
of FabL has not been reported, although it has been crystallized (Kim, Park et al. 2007).
Triclosan Resistance of FabV—Early in our work we found that expression of FabV in E.
coli imparted resistance to triclosan (Figure 3.2B). A similar picture was seen in an E.
coli in vitro fatty acid synthesis system in which the inactivated mutant FabI was replaced
with FabV (Figure 3.12) or when an in vitro fatty acid synthesis system prepared from V.
cholerae cells was treated with triclosan (data not shown). Moreover, growth of the wild
type V. cholerae strain ATCC 14547 was 20-fold more resistant to triclosan than was the
wild type E. coli strain MG1655 (Figure 3.13). These observations argued that FabV
might be significantly more resistant to triclosan than is E. coli FabI. Two modes of
triclosan inhibition of enoyl-ACP reductases have been observed. Some triclosansensitive enoyl-ACP reductases, such as FabI, are irreversibly inhibited by formation of a
dead-end reductase-NAD+-triclosan ternary complex, whereas other reductases such as
FabL show reversible inhibition without formation of a ternary complex. Moreover,
under the usual assay conditions triclosan is a slow binding inhibitor of FabI-type
enzymes because the reductase-NAD+ complex must be formed before the inhibitor can
bind. Triclosan inhibition of FabV is very weak (Figure 3.9). However, the degree of
FabV inhibition increased as the reaction proceeds indicating that a product, presumably
NAD+, may be involved in the inhibition. Indeed, triclosan inhibition of FabV was
potentiated by preincubation with NAD+ (Figure 3.9). However, even in the presence of
NAD+, triclosan was not a potent inhibitor of FabV.
50
3.5 DISCUSSION
We have identified a new enoyl-ACP reductase isoform encoded by V. cholerae. The
lack of sequence alignment with known enoyl-ACP reductases together with its atypical
active site and much greater size indicates that the V. cholerae enzyme should be
considered a member of a new class of enoyl-ACP reductases having homologues in both
closely and distantly related species of bacteria. We have named this fourth enoyl-ACP
reductase isoform, FabV for Vibrio, a species in which this is the only enoyl-ACP
reductase now predictable by sequence alignment. Like its FabI and FabL functional
homologues, it is a member of the SDR superfamily. This very large family of NAD(P)dependent oxidoreductases shows a significantly conserved structure despite little
sequence homology (15–30%) among members. The largely conserved SDR folding
pattern allows specific sequence motifs to be assigned, with those for the coenzymebinding and active site regions being the most definitive. A recent bioinformatic analysis
of the SDR superfamily placed the enzymes into five families (Persson, Kallberg et al.
2003). One of these families (called divergent) is composed of the FabI-type enoyl-ACP
reductases of bacteria and plants (Persson, Kallberg et al. 2003). FabV does not fall
cleanly into this or any of the other four families. FabV is 60% larger than the typical
SDR family member (which are generally about 250 residues in length) and the spacing
between the putative FabV active site tyrosine and lysine residues is eight residues, two
more than FabI and FabL and one more than the maximum reported for other SDR
proteins. Conversely the coenzyme-binding site of FabV has the classical Rossman fold
51
motif like that of FabL, whereas the FabI coenzyme-binding fold departs from that motif.
Persson and co-workers (Persson, Kallberg et al. 2003) have reported that the coenzyme
preference for the SDR enzymes can be predicted with >93% accuracy based on the
presence or absence of key residues. The very strong preference of FabV for NADH over
NADPH fits the predictive rules of Persson and co-workers (Persson, Kallberg et al.
2003). These rules also correctly predict the coenzyme preferences of B. subtilis FabL
(NADPH strongly preferred) (Heath, Su et al. 2000) and E. coli FabI (little or no
preference) (Bergler, Fuchsbichler et al. 1996).
In vivo and in vitro assays demonstrate that expression of FabV restores the otherwise
defective de novo fatty acid synthesis of the E. coli fabI392(Ts) mutant at the
nonpermissive temperature to normal (Figures 3.2B and 3.4). Because FabI is the sole
enoyl-ACP reductase of E. coli and is known to reduce enoyl-ACPs of acyl chain length
from C2 to C18 (Heath and Rock 1995; Heath, Su et al. 2000), the complementation data
indicate that FabV is active with this range of acyl chain length substrates. Moreover,
expression of FabV confers triclosan resistance to E. coli (Figures 3.2B and 3.13) as
previously seen for FabK and FabL. The resistance imparted by FabV is also observed in
cell extracts and for the purified enzyme. Therefore, FabV is the first intrinsically
triclosan-resistant enoyl-ACP reductase found in a Gram-negative bacterium. Indeed,
FabV shows little inhibition at triclosan concentrations approaching the solubility limit of
the compound. Moreover, expression of FabV from a single copy vector in E. coli made
the host more resistant to triclosan than was the V. cholerae strain from which the
encoding gene was derived. Two of the possible explanations for this unexpected result
are that V. cholerae may have a second target for triclosan, either another enoyl-ACP
52
reductase or a FAS II-independent target (McDonnell and Russell 1999; Escalada,
Russell et al. 2005) or that FabV expression is negatively regulated in V. cholerae, but
not in E. coli.
Triclosan is a very weak inhibitor of FabV even when inhibition is potentiated by
addition of NAD+. For example, FabL shows 50% inhibition by 12 μM triclosan (9),
whereas the same extent of inhibition of FabV requires 136 μM triclosan in the absence
of added NAD+ (Figure 3.9). Interpretation of the effects of NAD+ on triclosan inhibition
is complicated by the finding that NAD+ was a much stronger inhibitor of FabV than
triclosan (5 μM NAD+ inhibited similarly to 168 μM triclosan). This is not the case for
FabI. NAD+ does not inhibit FabI and indeed FabI fails to bind NAD+ unless triclosan is
present (Heath, Rubin et al. 1999; Ward, Holdgate et al. 1999; Heath, Su et al. 2000;
Sivaraman, Zwahlen et al. 2003). Triclosan increases the affinity of FabI for NAD+ by
1000-fold such that the inhibitory ternary complex can be formed (Heath, Rubin et al.
1999; Ward, Holdgate et al. 1999; Heath, Su et al. 2000; Sivaraman, Zwahlen et al.
2003). FabV may also form an inhibitory ternary complex. If so, the triclosan-mediated
increase in the affinity of FabV for NAD+ would be very modest relative to that seen with
E. coli FabI and would require much higher levels of triclosan. In the presence of NAD+
E. coli FabI is completely inhibited by 2 μM triclosan (Heath, Rubin et al. 1999), whereas
complete inhibition of FabV in the presence of NAD+ required about 300-fold higher
levels of triclosan (Figure 3.9). In any event triclosan is such a weak inhibitor of FabV
that further investigation of the mode of inhibition seems of little practical use. In
contrast to FabI and FabV, the reversible triclosan inhibition of FabL does not seem to
involve its interaction with NAD+ (Heath, Su et al. 2000).
53
The diversity of the bacterial enoyl-ACP reductases relative to the lack of structural and
mechanistic diversity seen in the other enzymes of the FAS II elongation cycle argues
that naturally occurring compounds exist that selectively inhibit one or another of these
enzymes. This hypothesis has recently been confirmed by the discoveries of natural
enoyl-ACP reductase inhibitors of fungal origin that specifically target FabI
(Cephalochromin) (Zheng, Sohn et al. 2007) and FabK (Atromentin and Leucomelone)
(Zheng, Sohn et al. 2006).
54
3.6 TABLES AND FIGURES
Figure 3.1 The elongation cycle of fatty acid biosynthesis. Each elongation cycle is
initiated by the condensation of malonyl-ACP with acyl-ACP carried out by one of the 3ketoacyl-ACP synthases. The second step is the reduction of 3-ketoester by 3-ketoacylACP reductase followed by the dehydratation of the resulting 3-D-hydroxyacyl-ACP to
the trans-2 unsaturated acyl-ACP. This substrate that at the C4 stage is called crotonylACP is reduced by enoyl-ACP reductase such as FabV, FabI, FabL, or FabK to generate
an acyl-ACP two carbons longer than that the original acyl-ACP substrate.
55
Figure 3.2 The cosmid complements growth of an E. coli fabI(Ts) strain and
also imparts resistance to triclosan. Panel A shows the growth of the E. coli fabI(Ts)
mutant strain PMT03 at either the permissive temperature of 30 °C or the non-permissive
temperature of 42 °C when transformed with either the fabV cosmid pMT18 or the vector
pCC1fos. Panel B shows the growth of the E. coli fabI(Ts) mutant strain PMT03 at the
permissive temperature of 30 °C in the presence or absence of 2 µg/ml triclosan. The
medium used was LB medium containing 12 µg/ml chloramphenicol.
56
Figure 3.3 Alignments of FabV with FabI and FabL. The V. cholerae FabV sequence
(top line) is that determined in this work, whereas the FabI (middle line) and FabL
(bottom line) sequences are from E. coli and B. subtilis, respectively. The active site
tyrosine and lysine residues are marked with asterisks.
57
Figure 3.4 Assay of complementation in vivo. Cultures of the E. coli fabI(Ts) strain
JP1111 carrying either the vector or the complementing cosmid or the wild type (WT)
strain MG1655 were labeled with [1-14C]acetate followed by extraction of the cellular
phospholipid and analysis by TLC. The presence or absence of the complementing
cosmid is denoted by plus or minus signs, respectively. An autoradiogram of the TLC
plate is shown. The phospholipids are phosphatidylethanolamine (PE),
phosphatidylglycerol (PG), and cardiolipin (CL).
58
Figure 3.5 Growth of the E. coli fabI(Ts) strain transformed with plasmids
encoding three different forms of FabV. Each version of the fabV gene was cloned into
the arabinose-inducible low copy vector, pBAD322, and these plasmids were transformed
into the fabI(Ts) strain JP1111. Serial 10-fold dilutions of each culture were spotted
(from top to bottom) onto plates of LB medium supplemented with 0.2% glucose to
repress the arabinose promoter. The plates were incubated at 42 °C.
59
Figure 3.6 Purification of FabV. The gel of panel A shows the purification of the Cterminal His6-tagged protein, whereas the two gels of panel B show the purification of
the native protein. In the gel of panel A fractions from the nickel-nitrilotriacetic acid
purification were loaded. The fractions loaded were: CE, crude extract of an induced
culture 1, crude extract of an induced culture 2; FT1 and FT2, flow-through fractions 1
and 2; W1 and W2, wash fractions 1 and 2; and E, combined His-tagged FabV fractions 1
and 2. Mk denotes the protein molecular weight markers. The left gel of panel B shows
the purification of native FabV through a DEAE column in which the bulk of the enzyme
eluted with 0.4 M NaCl, whereas the right gel of panel B shows further purification of the
DEAE enzyme by elution from a Blue Sepharose column. The conditions were as given
under ―Experimental Procedures.‖ The right gel of panel B shows the purified FabV after
Blue Sepharose chromatography. The fractions loaded were: CE1 and CE2, which are
crude extracts of cultures induced with 1.5 or 0.75mM isopropyl-Dthiogalactopyranoside, respectively. The flow-through (FT) fraction and the fractions
eluted with 400 or 700 mM NaCl (designated 400 and 700) are also shown. Other
abbreviations are as in panel A. The gels were 8% SDS-PAGE gels stained with
Coomassie Blue. Panel C shows the elution behavior of FabV (determined by
absorbance, enzyme activity, and gel mobility) on a Superdex 200HR 10/30 column. The
standard proteins were chymotrypsinogen (CT), ovalbumin (OA), bovine serum albumin
(BSA), aldolase (ALD), and ferritin (Fer).
60
Figure 3.7 Kinetic analysis of the native FabV enoyl-ACP reductase. The
NADHconsumption per minute per pg of protein was plotted against varying
concentrations of NADH (panel A) or crotonyl-ACP (panel B). The Km and Vmax
values were determined. The Km value for NADH was 53µM, whereas that for crotonylACP was 195 µM. The crotonyl-ACP concentration in panel A was 80 µM, whereas the
NADH concentration in panel B was 150 µM.
61
Figure 3.8 Activity and specificity of FabV. Panel A shows the relative activities of the
native (solid squares) and C-terminal His6-tagged (open triangles) forms of FabV. The
control lacking crotonyl-CoA is also shown (X-X). Panel B shows the relative activities
of FabV with 150 µM NADH (solid squares) or 150 µM NADPH (open triangles). The
NADH control lacking crotonyl-CoA is also shown (X-X).
62
Table 3.1 The kinetic parameters of FabV. The catalytic efficiency of each enzyme was
determined under pseudo first-order conditions with NADH in excess and crotonyl-CoA
(or crotonyl-ACP) concentrations under Km values (100 and 15 µM, respectively). Under
these conditions, the first-order rate constant kobs was determined over time and catalytic
efficiency was determined as kobs/[ET] = Kcat/Km. The enzyme concentrations were
2.61 pM for native FabV and 25 nM for the C-terminal His6-tagged (C-tag) FabV. The
C-terminal-tagged FabV was studied only with crotonyl-CoA and NADH. The large
standard deviation seen with NADPH as the substrate was due to the very low activities
observed.
63
Figure 3.9 Effect of triclosan on FabV activity. Panel A shows the effects of triclosan
addition to the standard reaction mixture containing native FabV, NADH, and crotonylCoA. In panel B native FabV was incubated in the presence or absence of 5 µM NAD+
and triclosan as shown prior to initiation of the enoyl reductase reaction by addition of
200 µM crotonyl-CoA.
64
Figure 3.10 Comparisons of the genomic sequence V. cholerae El Tor N16961 with the
V. cholerae ATCC 14547 sequence and those of other Vibrio species. Panel A: The DNA
segment common to the complementing subclones derived from the V. cholerae cosmid.
The map at the top of the panel is a section of the pathogenic V. cholerae El Tor N16961
genome sequence (chromosome 1) and was obtained from the TIGR database (11).
VC1738 and VC1739 are ORFs annotated as coding for hypothetical proteins. The
arrows below the region map represent the 4 subclone plasmids that complemented the
fabI(Ts) mutant strains and also conferred resistance to triclosan. Panel B: The putative
frameshift mutation caused by the adenine omitted in the pathogenic V. cholerae genome
sequence. The arrow indicates the nucleotide missing in the El Tor N16961 sequence
(which is a guanine in the V. vulnificus genome).
65
Figure 3.11 Assay of complementation in vitro. The extreme left column shows the fatty
acid synthetic activity of an extract of the wild type (WT) E. coli strain MG1655 whereas
the extreme right column shows the fatty acid synthetic activity of an extract of the
fabI(Ts) strain JP1111 supplemented with 500 nM FabV carrying a C-terminal
hexahistidine tag. The two central columns are duplicate incubations of the fabI(Ts)
extract without added FabV.
66
Figure 3.12 FabV imparts triclosan resistance to fatty acid synthesis by E. coli cell
extracts. Radioactivity from [2-14C] malonyl-CoA incorporated into fatty acids in the
presence of various concentrations of triclosan was measured in counts per minute with a
liquid scintillation counter. In panel A the enzyme preparation was from wild-type E. coli
whereas in panel B the preparation was from the fabI(Ts) strain JP1111 which was either
supplemented with 500 nM FabV carrying a C-terminal hexahistidine tag or left without
supplementation. The FabV containing samples also received triclosan at the
concentrations shown.
67
Figure 3.13 V. cholerae is less sensitive to triclosan than E. coli by 20-fold. V. cholerae
and E. coli were grown on LB in the absence or presence of increasing amounts of
triclosan. 0.4 µg/mL of triclosan is necessary to observe an effect on V. cholerae growth
(A), while it only takes 0.02 µg/mL of triclosan to affect E. coli growth (B) (20-fold
difference). As high as 10 µg/mL of triclosan is necessary to observe a slight effect on the
E. coli fabI(Ts) mutant (PMT03) complemented with the fabV cosmid. FabV seems to
confer a greater resistance to triclosan when expressed in E.coli than when present in V.
cholerae, suggesting either another triclosan target or the presence of a negative regulator
of FabV in V. cholerae. A more efficient efflux pump in E. coli is also a possibility.
Because a fabV homologue is also predicted, by sequence homology, to be present in the
pathogenic agent responsible for the plague (Yersinia pestis), we also tested that strain for
growth on triclosan (data not shown). Y. pestis showed a Minimum Inhibitory
Concentration (MIC) even higher than V. cholerae (4 µg/mL versus 0.8 µg/mL).
68
CHAPTER 4
A NEW ENOYL-ACYL CARRIER PROTEIN REDUCTASE ISOZYME, FABS
4.1 ABSTRACT
Enoyl-acyl carrier protein reductases (ENRs) are essential enzymes that catalyze the last
step of the fatty acid elongation cycle. Four different isozymes are known, one of which
is FabV of Vibrio cholerae. We report a new V. cholerae ENR, FabS, which unlike
FabV, is sensitive to the antibacterial, triclosan.
4.2 A NOVEL TRICLOSAN-SENSITIVE VIBRIO CHOLERAE ENOYL-ACYL
CARRIER PROTEIN REDUCTASE
The dissociated (type II) fatty acid synthase system (type II) is an essential pathway
composed of discrete proteins that each catalyze a single step of the pathway that is
thought a good target for new antibacterials. Fatty acids are synthesized in two carbon
increments by a repetitive cycle (called the elongation cycle) of condensation, reduction,
and dehydration with the final reduction step being catalyzed by the enoyl-acyl carrier
protein (ACP) reductase (ENR) which is the most diverse of the fatty acid synthetic
proteins (Massengo-Tiasse and Cronan 2009). The only irreversible reaction of the
69
elongation cycle is the 3-ketoacyl-ACP synthase step that initiates each cycle (often
called the condensation step, although this is a misnomer), the other reactions are freely
reversible. However, unlike the first reduction and the dehydration steps of the cycle, the
equilibrium of the ENR is strongly in favor of its product which serves to pull each cycle
to completion (Heath and Rock 1995). FabI, the ENR of E. coli is also the specific target
of the broad-spectrum antibacterial agent, triclosan (Massengo-Tiasse and Cronan 2009).
However, several other ENR isozymes, FabL (Heath, Su et al. 2000), FabK (Marrakchi,
Dewolf et al. 2003) and FabV (Massengo-Tiasse and Cronan 2008) were discovered
through their resistance to triclosan, although the enzymes show varying degrees of
resistance. In prior work we found that expression Vibrio cholerae FabV imparted very
high levels of triclosan resistance to E. coli whereas V. cholerae itself was only modestly
resistant to the microbiocide (Massengo-Tiasse and Cronan 2008). In contrast
Pseudomonas aeruginosa, which contains both FabV and FabI, is extremely resistant to
triclosan (Zhu, Lin et al. 2010). Genetic analysis in P. aeruginosa showed that FabV is
responsible for the resistant phenotype and since FabV and not FabI is required for rapid
growth, FabV is the dominant ENR of this bacterium (Zhu, Lin et al. 2010). From these
data it seemed likely that FabV might be a minor ENR in V. cholerae and that the bulk of
the enoyl-ACP reduction in this bacterium is catalyzed by one or more triclosan-sensitive
ENRs. Since FabI is the only known triclosan-sensitive ENR, we have addressed the
question of triclosan-sensitive ENRs in V. cholerae. The methods and strains used were
those of the prior Chapter 3 (Massengo-Tiasse and Cronan 2008) and any modifications
will be given in context. The V. cholerae bv. albensis ATCC14547 strain is an
environmental isolate.
70
Indirect evidence for a triclosan sensitive ENR was obtained by the finding that, although
V. cholerae is relatively resistant to triclosan (Massengo-Tiasse and Cronan 2008), sublethal doses of the biocide altered the fatty acid composition of the bacterium (data not
shown). Encouraged by this finding we constructed a new cosmid library having a
greater degree of genome coverage than our previous library and again screened for ENR
encoding cosmids by complementation of an E. coli fabI(Ts) strain. The strains carrying
the complementing cosmids were then tested for triclosan resistance (Figure 4.1). We
obtained two sets of cosmids, each contained an array of overlapping genome segments.
All four cosmids that gave triclosan resistant transformants contained the fabV sequence
and thus FabV appears to be the sole triclosan-resistant V. cholerae ENR. The four
complementing clones that failed to impart triclosan resistance remained sensitive even
when the cosmid copy numbers were increased by induction with arabinose suggesting
that the encoded ENR was highly sensitive to the inhibitor. Subcloning of the genes in the
overlapping region of these cosmids (Figure 4.1) showed that all carried the V. cholerae
ATCC14547 locus VCA001683 which encodes a protein 100% identical to the VC2093
locus of the fully sequenced V. cholerae El Tor genome. These loci encode a ―conserved
hypothetical protein‖ annotated as a putative member of the Nif-3 protein superfamily
(Pfam01784) that we call FabS. A C-terminally hexahistidine-tagged version of FabS was
expressed in E. coli and purified to homogeneity by Ni-chelate chromatography (Figure
4.2). To test for complementation in vitro we added purified FabS to an in vitro fatty acid
biosynthesis system partially purified from extracts of cells of the fabI(Ts) strain
(PMT03) grown at the permissive temperature and then shifted to the non-permissive
temperature. These manipulations result in decreased fatty acid synthesis due to an ENR
71
deficiency that can be restored by addition of a purified ENR (Massengo-Tiasse and
Cronan 2008). Indeed addition of purified FabS to such extracts gave increased
radiolabeled fatty acid synthesis, but only when NADPH was the electron donor. These
data indicated that FabS uses NADPH in vivo and can utilize acyl carrier protein
substrates.
The purified protein was found to reduce the model substrate, crotonyl-CoA, using either
NADH or NADPH as the hydrogen donor (all known bacterial ENRs reduce crotonylCoA) (Figure 4.3). However, as the NADH reaction progressed it slowed and at late
times showed a decrease in the yield of oxidized cofactor suggesting reversal of the
reaction. Moreover, high concentrations of NADH (e.g., 600 µM) completely blocked
the reductase activity of FabS (data not shown) perhaps due to contaminating NAD.
Thus the lack of activity with NADH seen in the in vitro complementation experiments
(Figure 4.2) can probably be attributed to NAD inhibition of the enzyme. As expected the
FabS ENR activity was sensitive to triclosan with 2.5 µg/ml giving essentially complete
inhibition. This triclosan concentration is sufficient to block growth of Vibrio cholerae
(Massengo-Tiasse and Cronan 2008). It should be noted that under the assay conditions
chosen FabS was almost 10-fold less active in crotonyl-CoA reduction than FabV (data
not shown) consistent with its lower activity in the in vitro complementation system. The
results obtained with the model compound were reflected in the relative abilities of FabS
and FabV to rescue fatty acid synthesis in ENR deficient cells or cell extracts. We also
tested the effects of FabS expression on the fatty acid composition of cultures of a
fabI(Ts) E. coli strain carrying the fabS cosmid that were grown at 30° C and shifted to
72
42° C for 30 min and found that significantly more 18.1 and less 16.0 fatty acid species
were produced relative to parallel cultures lacking the cosmid (data not shown).
The original member of the Nif-3 (Pfam01784) protein family was identified in yeast by
two-hybrid screening for proteins that interacted with the NGG1p transcription factor
(Martens, Genereaux et al. 1996), the functional significance of which remains unclear.
This is particularly so since the subsequent report that Nif-3 is an abundant mitochondrial
protein (Reinders, Zahedi et al. 2006). Although members of the Pfam01784 protein
family are widely distributed in all three domains of life and, no discrete biological
function as been demonstrated for any member. Although FabS lacks the canonical
Rossman fold nucleotide binding site, as do a number of other proteins that bind pyridine
and flavin nucleotides (Rondeau, Tete-Favier et al. 1992; Boyd, Endrizzi et al. 2011), a
putative nucleotide binding site has been proposed for one of the Pfam01784 proteins of
known structure, Bacillus cereus YqfO (Godsey, Minasov et al. 2007), whereas a large
unidentified ligand in the analogous location is found in another Pfam01784 protein,
Staphylococcus aureus SA1388 that could be NADPH. The UV-visible spectrum of
FabS indicates that it is not a flavoprotein (data not shown). The putative nucleotidebinding site is supported by analysis of the FabS sequence using the NCBI Conserved
Domains database which detected several conserved domains (Figure 4.4). Near the Nterminal end of FabS sequences are found that align with the N-terminal end of the
NADP binding site of the Medium Chain Dehydrogenase Reductase superfamily (MDR)
(domain cd08250). This domain is related to the cd08270 domain found in FabI as well
as to domain cd08290 of the FabK ENR and cd08292 plus Pfam12242 of the FabL ENR.
Note that cd08929 and cd08290 are specifically defined as domains of 2-enoyl thioester
73
reductase like proteins, catalyzing the NADPH-dependent reduction of trans-2-enoyl
thioesters to acyl-thioesters in fatty acid synthesis. All of these domains belong to the
cl14614 domain family. Two enoyl-CoA hydratase domains (PRK12478 and
PRK06143), also found in FabK and FabI, align with the center of FabS and are members
of the crotonase-like superfamily (cl11392). Thus FabS seems to be an interesting
mélange of sequences from the SDR (short chain dehydrogenases/reductases)
superfamily (which includes all of the ENRs except FabK) and the MDR superfamily.
This is not unexpected because the SDR and MDR families are thought to have arisen
from a common ancestor (Jornvall, Hedlund et al. 2010). Some MDRs are zinc enzymes
whereas no SDR metalloenzymes are known (Jornvall, Hedlund et al. 2010). Some of
the Pfam01784 proteins of known crystal structure (Saikatendu, Zhang et al. 2006;
Godsey, Minasov et al. 2007) have bound metal ions whereas others do not. We tested
the effects of the chelator, ethylenediaminetetraacetic acid, on the ENR activity of FabS
and found that the reaction was only slightly inhibited by 10 mM
ethylenediaminetetraacetic acid (data not shown) suggesting that either FabS is not a
metalloprotein or has metals that are well protected from solvent. The mosaic nature of
FabS undoubtedly accounts for its lack of annotation. In this regard it is noteworthy that
FabK was annotated as a nitropropane oxygenase involved in nitrogen metabolism. Not
all FabK homologues carry the ENR activity, as previously tested in Bacillus subtilus,
Staphylococcus aureus and Pseudomonas aeruginosa (Marrakchi, Dewolf et al. 2003;
Zhu, Lin et al. 2010). Likewise, it seems very unlikely that all Pfam01784 proteins will
be ENRs. Indeed, we tested putative homologues of FabS found in Vibrio fisheri and a
close Vibrio relative, Photobacterium profundum, for fabI(Ts) complementation and both
74
failed to complement. Moreover, E. coli has YbgI, a canonical Pfam01784 protein, but
the sole ENR detected in this bacterium by biochemical and genetic means is FabI
(Bergler, Hogenauer et al. 1992; Bergler, Wallner et al. 1994; Bergler, Fuchsbichler et al.
1996). The SDR and MDR protein families have an enormous variety of substrates and
we expect that similar substrate diversity will be true of the Pfam01784 proteins.
75
4.3 FIGURES
Figure 4.1 Restoration of growth and fatty acid synthesis to the fabI(Ts) strain (PMT 03)
by fabS expression. The plates in the top row (A, B, C) show the phenotypes of strain
PMT03 carrying the original cosmids isolated by complementation. The phenotypes at
the permissive (30o C) and non-permissive (42o C) temperatures and the presence of 2
μg/ml of triclosan (Tcl) are given. The plates of the lower row show the phenotypes of
strain PMT03 expressing either the fabS gene (plate E) or an irrelevant gene from the
same cosmid (plate D). The genes were expressed under control of the E. coli araBAD
promoter of pBAD322K in the presence of 0.2% arabinose. In the autoradiogram of
panel F, we show radiolabeled phospholipids from exponentially growing cultures
expressing either a control plasmid or fabS. Cells were shifted from 30o C to 42o C. After
~5 min at 42o C [1-14C] acetate was added and 40 min later the cellular phospholipids
were extracted and analyzed as previously describe.
76
Figure 4.2 Purification of FabS and its ability to restore in vitro fatty acid biosynthesis in
the presence of NADPH. Panel A: Lane 1, molecular mass standards in kDa and lane 2,
purified FabS. Panel B: The ability of either purified FabS (685 nM) or FabV to restore
de novo fatty acid biosynthesis was measured by addition of each protein to an in vitro
fatty acid biosynthesis system partially purified from extracts of cells of the fabI(Ts)
mutant (PMT03) grown at the permissive temperature and then shifted to the nonpermissive temperature. The assays were done for 75 min at 37°C as described except
that the reaction volume was 40 µL and 10 µCi of [14C] malonyl-CoA was added. The
reduced pyridine nucleotide concentration was 150 µM.
77
Figure 4.3 Crotonyl-CoA reductase activity of FabS and its inhibition by triclosan (Tcl).
Panels A and B show the time course of FabS reduction of crotonyl-CoA over short
(panel A) and extended (panel B) time scales. The gray curve was assayed NADPH
whereas the black curve was assayed with NADH. Panel C shows the effects of triclosan
on FabS activity in the presence of NADPH. The concentrations of reduced pyridine
nucleotide, crotonyl-CoA and FabS were 150 µM, 300 µM and 15 nM, respectively. The
buffer was 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl and the
assays were run at room temperature.
78
Figure 4.4 The sequence of FabS from V. cholerae albensis. The bold letters on the top
line are the sequence that aligns with the NADP binding site of the MDR superfamily.
The bold italicized letters of the second line denote the sequence that aligns with a set of
NADH dependent reductases whereas the underlined sequence of the third line aligns
with enzymes known to use enoyl thioester substrates.
79
CHAPTER 5
CONCLUSIONS
5.1 SUMMARY
The principal aim of this study was to uncover a new class of enoyl-acyl carrier protein
(ACP) reductase in Vibrio cholerae, and explore the diversity of this key fatty acid
biosynthesis enzyme.
As described in the introduction, the original enoyl-ACP reductase (ENR), FabI, was first
discovered in E. coli (Egan and Russell 1973; Bergler, Wallner et al. 1994; Bergler,
Fuchsbichler et al. 1996). Some of the characteristic features of FabI are that FabI is an
NAD(P)H-dependent reductase and it is the specific target of the commonly used
antibacterial agent, triclosan. Because of FabI’s wide distribution across bacterial species,
it was considered the only ENR, until the later discovery of two novel isozymes, the
NADPH-dependent FabL in Bacillus subtilus (Heath, Su et al. 2000) and the NADH
oxidase flavoprotein, FabK in Streptococcus pneumoniae (Marrakchi, Dewolf et al.
2003), both resistant to triclosan. Despite their difference in respect to their sensitivity to
triclosan and their cofactor specificity, FabI and FabL are related. FabK, on the other
hand, is structurally related to a 2-nitropropane dioxygenase (NPD). NPD enzymes are
normally known to catalyze oxidative denitrification of nitroalkanes.
80
The diversity of enoyl-ACP reductases made it difficult to accurately predict that a given
gene sequence would code for an active enoyl-ACP reductase (ENR).
Since no known ENR isoforms were detected in V. cholerae, we opted to functionally
complement an ENR deficient E.coli mutant with V. cholerae genomic libraries.
We first uncovered a novel ENR that we named FabV and demonstrated that this novel
enzyme showed resistance to triclosan, both in vivo and in vitro. We found that although
FabV belonged to the same superfamily as FabI and FabL, it was a significantly larger
protein and did not display the typical Y-(X)6-K motif displayed in the active site of
ENRs from this superfamily (Massengo-Tiasse and Cronan 2008). Instead, FabV
displayed a particular Y-(X)8-K motif, demonstrating that FabV did define a new class of
ENR. Predicted homologues of FabV were mostly found in gram-negative pathogens. We
confirmed that microorganisms expressing fabV are expected to be resistant to triclosan
as we observed with E. coli expressing fabV and a wild type Yersinia pestis strain.
Referring to our study, other laboratories have later confirmed our observations in
Pseudomonas aeruginosa (Zhu, Lin et al. 2010) and Burkholderia mallei (Lu and Tonge).
Interestingly, we found that despite having fabV, V. cholerae, although significantly more
resistant to triclosan than wild type E. coli, still displayed some sensitivity to the biocide.
We speculated either the presence of a fabV negative regulator only present in Vibrio
cholerae or the presence of another triclosan-sensitive ENR isozyme could explain this
inconsistency. Our search for a fabV regulator was ultimately not successful despite a
laborious effort.
The ideal way to check for the presence of another ENR in Vibrio cholerae (Vc) would
have been to check whether fabV was essential or not, by constructing a fabV knock-out
81
in V. cholerae in the presence of an inducible vector containing a functional replacement
ENR gene (fabI for example). If fabV were to be essential, meaning the only ENR present
in V. cholerae, then the organism would have been able to survive only upon induction of
the replacement ENR on the plasmid. My attempt was not successful. My failure to
obtain a fabV knock-out is attributed to a number of technical difficulties. Genetic
manipulations in Vibrio cholerae are known to be more challenging than in E. coli.
An indirect approach was to check whether the fatty acid profile of V. cholerae would be
affected by the presence of sub-lethal doses of triclosan. If the only ENR present in V.
cholerae was the triclosan-resistant FabV, we would not expect much change in the fatty
acid profile. If, on the other hand, growth with triclosan significantly affected the fatty
acid profile then the presence of a novel triclosan-sensitive ENR would be a reasonable
assumption. We in fact observed a significant effect of triclosan on the fatty acid profile
of V. cholerae and proceeded to screen another V. cholerae genomic library for
complementation of the ENR-deficient E. coli mutant. This time, we obtained a greater
coverage of the Vc genome. This led us to at least one more ENR candidates. Other
microorganisms are known to carry more than one active ENR: Bacillus subtilis (Heath,
Su et al. 2000), Pseudomonas aeruginosa (Zhu, Lin et al. 2010) and Burkholderia mallei
(Lu and Tonge 2010).
At this time, we have characterized FabS, the novel triclosan-sensitive ENR in Vibrio
cholerae. Its activity in vivo and in vitro is NADPH-dependent. FabS is neither a member
of the Short chain Dehydrogenase Reductase (SDR) superfamiy (to which FabI, FabL
and FabV belong), nor related to the 2-nitropropane dioxygenase (NPD) like FabK.
Hence FabS defines yet another class of ENRs.
82
It is interesting to note that some apparent homologues of the known ENR isoforms are in
fact not active. Besides fabI and fabL, Bacillus subtilis (Bs) was also predicted to carry a
fabK homologue. While BsfabI and BsfabL are in fact active ENR genes (Heath, Su et al.
2000), BsfabK failed to show any ENR activity. A similar observation was made for the
putative Staphyloccus aureus fabK (Marrakchi, Dewolf et al. 2003). Pseudomonas
aeruginosa was also predicted to have an active FabK protein. However, experimental
analysis of the Pseudomonas fabK homologues proved otherwise (Zhu, Lin et al. 2010).
Its fabV homologue, on the other hand, was active and solely responsible for the
bacterium’s resistance to triclosan (Zhu, Lin et al. 2010). Unlike the V. cholerae fabV, I
found that the Vibrio fischeri homologue failed to complement the E. coli fabI mutant. I
also tested the Vibrio fischeri homologue of fabS for complementation of the fabI mutant,
in the hope that fabS would be active. The Vibrio fischeri also failed to complement the
E. coli fabI mutant. Finally a putative fabS homologue from Photobacterium profundum,
a deep-sea -proteobacterium closely related to Vibrio species failed to complement. As
in the case of FabK, an active FabS-like ENR protein seems to be restricted to at best a
few microorganisms. The FabKs and FabS may be active, but in another reaction.
This apparent lack of homogeneity among the different classes of ENRs suggests that an
interesting evolution process could have led to the gain or loss of ENR activity for these
families of proteins. These evolutionary processes could describe a type of convergent
evolution involving the recruitment of conserved domains. A closer look at the conserved
domains in all the previously characterized and the novel ENRs may shed some light on
83
the relationship between those seemingly unrelated proteins, yet able to carry out the
same biological function (Figure 5.1).
5.2 PRELIMINARY DATA AND DISCUSSION
At this point, it is worth mentioning some additional preliminary data. The more recent V.
cholerae genomic library screening actually yielded two more potential ENR candidates.
One in which the protein has been tested in vitro and shows NADH-dependent enoylCoA reductase activity and sensitivity to triclosan. I will refer to this second Vibrio
cholerae triclosan-sensitive ENR candidate as Candidate 2 for now, since more work
needs to be done to identify the gene coding for the protein (see Figures 5.2, 5.3 and 5.4).
Although cloning a 3kb-complementing region into the pET101 expression vector
yielded the expression of an active protein, the exact sequence of the coding ORF
remains to be determined. This gene is particular in that it is located on the second
chromosome of Vibrio cholerae. Another complementing V. cholerae DNA fragment was
able to restore de novo phospholipid synthesis in the fabI mutant. I narrowed the
complementing region down to 14 Open Reading Frames (ORF). However, after
comparing all the conserved domains in each putative candidate ORF with the conserved
domains in all the ENR isoforms characterized so far, including FabV and FabS, one
ORF stood out as a potential ENR candidate. This ORF will have to be tested. This
candidate ORF aligns with a putative NAD(P) binding domain (cd08237) related to the
domains of a crotonyl-CoA reductase (cd08246) and an ENR-like domain (cd08249). It
84
also aligns with enoyl-CoA hydratase domains (COG1024 and PRK09245), both related
to the cl11392 domain found in FabI, FabK, FabS and Candidate 2. This putative ORF is
also linked to the crotonase-like superfamily (domain cl11392). This domain is also
present in FabI, FabK, FabS and Candidate 2. This superfamily includes a number of
enzymes playing important roles in fatty acid metabolism. If this ORF turns out to have
ENR activity, this study would have helped to establish a conserved domain pattern
associated with ENR activity.
There may also be physiological reasons explaining why different ENR isoforms are
necessary. A possible explanation given to rationalize the FabK activity is the anaerobic
metabolism of Streptococcus pneumoniae. S. pneumoniae is able to produce ATP mostly
via anaerobic glucose catabolism because of its lack of cytochromes. The NADH oxidase
activity of FabK may support glycolysis either via fatty acid biosynthesis or via substrateindependent NADH oxidation, as previously suggested (Marrakchi, Dewolf et al. 2003).
It is not clear whether, the FMN in FabK is required for enoyl-ACP reduction or if it is
used solely for substrate-independent NADH oxidase activity.
Like FabK, the purified Candidate 2 protein also shows a substrate-independent NADH
oxidase activity (Figure 5.2). It is not able to oxidize NADPH but is sensitive to triclosan
(Figure 5.3). The particularity of Candidate 2 is that it aligns with domains present in
members of the Na+/glutamate symporter (COG0786, TIGR00210) both related to
another Na+ transport domain (cl12073). This same domain is also present in FabS.
Moreover, a related domain also aligns with FabK, the glutamate synthase (GltS) FMNbinding domain (cd02808). Amazingly, this domain is found in the active Streptococcus
FabK but is absent in the inactive FabK-like Pseudomonas protein.
85
Very interestingly, for optimal growth, marine organisms like Vibrio species have a
particular requirement for Na+. Vibrio alginolyticus reportedly has a Na+-translocating
NADH-quinone reductase (Unemoto and Hayashi 1977; Tokuda and Unemoto 1982;
Hayashi, Nakayama et al. 2001). This respiration-driven Na+ pump is different however
from the proton pump in Klebsiella pneumonia in that it is a proton pump, occasionally
able to pump excess intracellular Na+ out (Krebs, Steuber et al. 1999).
What is also notable is that FabS aligns well with a domain present in Na+-pumping
NADH-quinone reductase of a number of marine bacteria (TIGR01939).
Bacteria-specific physiological requirements could help explain the, maybe necessary,
diversity in ENRs.
86
5.3 FIGURES
Figure 5.1 Four different superfamilies of proteins have a protein member with enoylACP reductase activity. FabI, FabL, FabK and FabS each contain conserved domains
related to at least 2 of the 4 superfamilies. FabI is a member of the SDR superfamily but
also contains a domain conserved in the Pfam01784 superfamily. FabL is a member of
the SDR superfamily but also contains a domain conserved in the TIM-phosphate binding
superfamily. FabK is a member of the TIM-barrel superfamily but also contains a
glutamate synthase domain (GltS). FabS is a member of the Pfam01784 superfamily but
also contains a conserved domain of the TIM-phosphate binding superfamily as well as a
GltS domain.
87
Figure 5.2 The Candidate 2 protein shows substrate-independent NADH oxidase activity
in the absence of the crotonyl-CoA.
88
Figure 5.3 The Candidate 2 protein is sensitive to triclosan.
89
Figure 5.4 Crotonyl-CoA reductase activity of the candidate 2 protein compared to
FabV.
90
CHAPTER 6
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105
CURRICULUM VITAE
Romelle Prisca Massengo-Tiassé
601 S. Goodwin Ave.
University of Illinois-Urbana-Champaign
Department of Microbiology
B103 Chemical and Life Sciences building
Urbana, Illinois 61801
________________________________________________________________________
Education:
University of Illinois, Urbana-Champaign, IL
PhD in Microbiology, 2011
Advisor: John Cronan, PhD
Dissertation title:
―Demonstration of novel enoyl-acyl carrier protein reductases in Vibrio cholerae‖
University of Wisconsin-Milwaukee, Milwaukee, WI
M.S in Biological Sciences, 2004
Advisor: Charles Wimpee, PhD
Master’s Thesis title:
―Evolutionary conservation of transcriptional activation of lux genes in the genus Vibrio‖
University Paris VI - Pierre et Marie Curie, Paris, FRANCE
B.S with honors, in Animal Physiology and Molecular Biology, minor in Biochemistry,
2001
________________________________________________________________________
Publications:
- Wier, Nyholm, Mandel, Massengo-Tiassé, Schaefer, Koroleva, Splinter-BonDurant,
Brown, Manzella, Snir, Almabrazi, Scheetz, Bonaldo, Casavant, Soares, Cronan, Reed,
Ruby, McFall-Ngai, Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2259-64.
―Transcriptional patterns in both host and bacterium underlie a daily rhythm of
anatomical and metabolic change in a beneficial symbiosis"
- Massengo-Tiassé and Cronan, Cell Mol Life Sci. 2009 May;66(9):1507-17.
"Diversity in enoyl-acyl carrier protein reductases"
- Massengo-Tiassé and Cronan, J Biol Chem. 2008 Jan 18;283(3):1308-16.
"Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase"
106
Select presentations:
- ―Characterization of a new enoyl-ACP reductase in the Vibrio species‖. First author
poster, with John Cronan, presented at the Molecular and Cellular Biology of Lipids
Gordon Research conference, 2007.
- ―Comparative analysis of lux operon regulatory regions in Vibrios‖. First author poster
presented at the 104th American Society of Microbiology General Meeting, with Aaron
Stephan and Charles Wimpee, 2004.
- ―Are lux operon organization and expression conserved in geographically isolated
strains of the same species?‖ Second author poster at the 54th Arctic Science Conference,
with Kevin Budsberg, Charles Wimpee and Joan Braddock. 2003.
________________________________________________________________________
Technical skills:
Performed microbiology experiments, biochemical and genetic analyses: protein overexpression and purification by ion exchange, affinity chromatography, gel filtration,
enzyme kinetic analyses; phospholipid, fatty acid extraction and analyses (Thin Layer
Chromatography, ESI and GC-MS); DNA purification and analysis (vector cloning;
cosmid library cloning; cell transformation, conjugation and P1 transduction; sitedirected mutagenesis and gene knock-out, DNA colony hybridization); Gene expression
analyses by DNA gel shift assays (DIG and P32 probe end-labeling and random primed
labeling), primer extension, gene fusions, RNA extraction and Northern blot analyses PhD candidate, Research Assistant, University of Illinois, Urbana-Champaign; M.S
candidate, University of Wisconsin-Milwaukee.
Web-based bioinformatics tools for comparative genomics and protein structure analyses
Bioscreen software
UNICORN software for AKTA purifier
Microsoft office (Excel, Powerpoint, Word)
________________________________________________________________________
Teaching experience:
- University of Illinois, Urbana-Champaign, IL
Teaching Assistant, Spring 2006, Fall 2007
Led weekly discussion sessions and helped prepare quiz questions for ―Molecular
Genetics‖.
- University of Wisconsin-Milwaukee, Milwaukee, WI
Teaching Assistant, Fall 2003 until Summer 2004
Led and coordinated all weekly discussion sessions. Composed, administered, and graded
quizzes as well as helped organize lecture sessions and determine final grades for
―Genetics‖.
107
- Teaching Assistant, Fall 2001 until Summer 2003
Prepared lecture notes and laboratory experiments. Administered and graded weekly
quizzes for ―Anatomy and Physiology II‖.
________________________________________________________________________
Academic Service:
Student Accessibility Center volunteer: Reading and recording textbooks for the
Alternative Text Format program, providing audio tapes of academic textbooks to
Blind/Visually Impaired students or physically-impaired students who cannot turn pages,
at the University of Wisconsin- Milwaukee (2001-2003). (Program Manager: Jean
Salzer).
Awards:
- Teachers Ranked as Excellent by Their Students for Molecular and Cellular
Biology/Life Sciences and College of Medicine Courses include students, academic
professionals and faculty, from the University of Illinois-Urbana-Champaign.
- Graduate research travel award, from the University of Wisconsin-Milwaukee.
Memberships:
American Society of Microbiology
International Federation of University Women
108