Download A Metabolic Node in Action: Chorismate

Critical Reviews in Microbiology, 27(2):75–131 (2001)
A Metabolic Node in Action: ChorismateUtilizing Enzymes in Microorganisms
F. Dosselaere and J. Vanderleyden*
Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20,
B-3001 Heverlee, Belgium
*
C o r r e s p o n d i n g a u t h o r . T e l e p h o n e : +3 2 – 1 6 3 2 1 6 3 1 . F a x : +3 2 – 1 6 3 2 1 9 6 6 . E-mail:
[email protected]
ABSTRACT: The shikimate pathway has been described as a metabolic tree with many branches
that led to the synthesis of an extensive range of products. This pathway is present only in
bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical
reactions of the pathway, significant differences exist in terms of organization and regulation. In
the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are
converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis
of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products
as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores.
Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase,
anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate
pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene
structures and regulation, nucleotide and protein sequences, protein structures, and reaction
mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways.
Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence
similarity, suggesting divergent evolution and commonality in reaction mechanisms. However,
other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction
capabilities.
I. INTRODUCTION
Bacteria, fungi, and plants are capable of
converting the carbohydrate precursors,
D -erythrose 4-phosphate and phosphoenolpyruvate, to shikimate and subsequently
to the dihydroaromatic compound chorismate. Shikimate and chorismate are the precursors for a wide range of compounds. This
pathway is known as the shikimate pathway
and has generated considerable interest because of its absence in animals, and hence
forms an attractive target for potential herbicides and antibiotics. In microorganisms, the
shikimate pathway is used to synthesize the
three proteinogenic aromatic amino acids,
that is, phenylalanine (Phe), tyrosine (Tyr),
and tryptophan (Trp); the folate coenzymes;
benzoid and naphtoid quinones; and a broad
range of mostly aromatic, secondary metabolites, including some siderophores. Although the shikimate pathway branches at
many points, chorismate is the last common
branch point for the above-mentioned compounds. Chorismate is converted by five distinct enzymes to prephenate, anthranilate,
aminodeoxychorismate, isochorismate, and
p-hydroxybenzoate, respectively (Figure 1).
These metabolites comprise the first committed intermediates in the biosynthesis of
1040-841X/01/$.50
© 2001 by CRC Press LLC
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FIGURE 1. Chorismate (in bold) as a precursor for (1) prephenate, (2) anthranilate, (3) aminodeoxychorismate,
(4) p-hydroxybenzoate, and (5) isochorismate. The enzymes involved are chorismate mutase (1), anthranilate
synthase (2), aminodeoxychorismate synthase (3), chorismate pyruvate-lyase (4), and isochorismate synthase (5). Chorismate is synthesized from phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P).
Phe and Tyr, Trp, folate, menaquinone and
the siderophore enterobactin, and ubiquinone,
respectively. The synthesis of these precursors is in most cases highly regulated. In
recent years, much effort has been given to
characterize the five chorismate-utilizing
enzymes. In this review, we focus on the
substantial progress that has been made in
the genetic and molecular characterization
of these enzymes and their reaction mechanisms. Comparisons of the chorismate-utilizing enzymes has shown that some of them
share sequence similarity, suggesting a common ancestor and commonality in reaction
mechanisms. The regulation of their activity
is discussed in view of how the metabolic
node functions in a microbial cell.
In plants, thousands of primary and secondary aromatic compounds, which play a
76
role in plant growth, development, and defense, are synthesized via the shikimate
pathway. The flow through the shikimate
pathway accounts for up to 20% of the photosynthetically fixed carbon in plants, most
of which is shuttled through Phe and Tyr to
generate abundant phenylpropanoid metabolites. The complex demand for aromatic
secondary metabolites in specific cell types
and in response to multiple environmental
stimuli suggests that the regulation of Phe
and Tyr biosynthesis in plants may differ
fundamentally from the regulation observed
in microorganisms. Due to these differences, we confine this review to microorganisms, and the reader interested in the
molecular organization of the shikimate
pathway in plants is directed to other reviews.1-6
For this review, DNA and protein sequences have been analyzed using BLAST7
and ClustalW.8 The available completed genomes sequences were additionally analyzed
using COG9 and WIT.10 It has to be mentioned that some annotations of published
genome sequences appear not to be correct.
The crystal structures mentioned in the text
can be consulted at http://oca.ebi.ac.uk/ using the corresponding protein database entry
codes.
II. CHORISMATE MUTASE
A. Introduction
Chorismate mutase (CM, EC 5.4.99.5)
is the first enzyme of the branch of the pathway leading to Phe and Tyr and catalyzes
the intramolecular 3,3-sigmatrophic rearrangement of the enolpyruvyl side chain of
chorismate to form prephenate. CM is a rare
example of an enzyme that facilitates a pericyclic process known as a Claisen rearrangement. It is found in bacteria, fungi, and
plants.4 The biology and the unique mechanism have led to extensive study of the
chorismate-prephenate rearrangement over
the last 3 decades.2,4,11 Here, we discuss the
recent work that elucidated the enzyme structure, reaction mechanism, and regulation at
the molecular level.
B. Structural Organization and
Regulation of Chorismate Mutase
and the Encoding Genes
Based on their protein structure, CMs
can be divided into two major classes: the
AroH class, which contains CMs with a trimeric pseudo α/β-barrel structure, and the
AroQ class that groups mostly dimeric, allhelix-bundle proteins. Different isoenzymes
can be found in microorganisms, which dif-
fer in their structural organization and allosteric regulation and provide alternative routes
for the biosynthesis of Phe and Tyr (Figure
2).4 Despite the many CMs that have been
separated and purified, until recently only a
few genes encoding CMs were cloned and
sequenced. With the explosion, since 1995,
of primary sequence information available
from genome sequencing of microorganisms,
many genes encoding putative CMs have
been identified. The different CM isoenzymes
comprise a set of functionally related proteins that show little sequence similarity to
each other. Recently, a new nomenclature
for the CM isoenzymes belonging to the
AroQ class has been proposed.12,13 The different classes are AroQp, AroQt, AroQd,
AroQf, and AroQr, where the subscript indicates whether the CM is fused to prephenate
dehydratase, prephenate dehydrogenase,
3-deoxy- D -arabinoheptulosonate-7-phosphate (DAHP) synthase, or is monofunctional and either unregulated or allosterically regulated, respectively.
1. AroQp or Chorismate Mutase
Fused to Prephenate Dehydratase
CM:prephenate dehydratase, or P-protein,
is a bifunctional protein, catalyzing two consecutive steps in Phe biosynthesis (Figure 2).
The CM domain catalyzes the formation of
prephenate, which is transformed into
phenylpyruvate by the action of the prephenate
dehydratase domain.14 The P-protein is encoded by aroQp.pheA and has been isolated
and characterized from Escherichia coli,14-16
Erwinia herbicola,17 Pseudomonas stutzeri,18
Xanthomonas campestris,12 and Buchnera
aphidicola.19 The Es. coli aroQp.pheA gene
product comprises 386 amino acids and has a
molecular mass of 43.1 kDa. The aroQp.pheA
gene is transcriptionally regulated by Phemediated attenuation. Upstream of the
aroQp.pheA gene, three putative mRNA stem-
77
FIGURE 2. Different pathways for the biosynthesis of L-Phe (6) and L-Tyr (7) in microorganisms. Chorismate
(1) is converted to prephenate (2) by the different CM isoenzymes, leading to either phenylpyruvate (3),
L-arogenate (4), or 4-hydroxyphenylpyruvate (5).
78
loop structures and a pheL leader region, encoding a 15-residue Phe-rich leader peptide,
can be found.20-22
The Es. coli native P-protein is a
homodimer that is allosterically retro-inhibited by Phe. Binding of Phe causes significant changes in the conformation of the enzyme and favors a shift in the aggregation of
the enzyme from an active dimer to less
active tetrameric or octameric species.23 The
activity of both CM and prephenate dehydratase is reduced by Phe with 55 and 85%,
respectively.14 Using genetically engineered
fragments of AroQp.PheA, it was shown that
the enzyme consists of a CM domain (CM-P
domain; residues 1–109), a prephenate dehydratase domain (residues 101–285) and a
regulatory domain (residues 286–386).24-26
Similar results for attenuational regulation of the aroQp.pheA gene and the structural organization of its product have been
found in Er. herbicola.17,22 In Ps. stutzeri the
aroQp.pheA gene appears to be organized in
a mixed-function supraoperon.27 Alternative
stem-loop structures between serC and
aroQp.pheA genes of Ps. stutzeri have been
found and suggest regulatory mechanisms at
the level of translation initiation.28 The Xa.
campestris aroQp.pheA gene probably forms
a transcriptional unit with the upstream serC
gene and encodes for a CM that possesses a
40-residue amino-terminal extension that is
Lys-rich and that is absent in all of the AroQp
homologs known at present.12
Sequence alignment with other Phe-binding proteins, such as monofunctional
prephenate dehydratases and Phe hydroxylases, identified two highly conserved sequences, GA[L,V] and ESRP, in the regulatory domain. Mutations in these regions
induced to different extent a reduced retroinhibition of the Es. coli CM activity and a
decrease in the Phe-binding capacity.29,30 The
P-protein of the aphid endosymbiont Bu.
aphidicola shows changes in the ESRP sequence of the regulatory domain. A consti-
tutive expression of the aroQp.pheA gene
and loss of the allosteric regulation by Phe
of the P-protein suggests an overproduction
of Phe, which complements dietary deficiencies of the aphid hosts.19
Similar aroQp.pheA genes, encoding putative P-proteins, can also be found in the
genome sequences of Thermotoga maritima,31
Haemophilus influenzae,32 Aquifex aeolicus,33
Buchnera sp. APS,34 Campylobacter jejuni,35
different Neisseria spp.,36,37 Vibrio cholerae,38
Xylella fastidiosa,39 Pasteurala multocida,40
and Pseudomonas aeruginosa.41 Alignment
of these putative CM:prephenate dehydratases
shows the conservation of many residues
throughout the whole amino acid sequence,
including the Phe-binding sites. In the CM-P
domain, the conserved amino acids all reside
in the N-terminal part (residues 1–60), which
are also conserved in the CM-T amino acid
sequence (see below).
2. AroQt or Chorismate Mutase
Fused to Prephenate
Dehydrogenase
CM:prephenate dehydrogenase, or T-protein, is involved in the biosynthesis of Tyr.
The prephenate dehydrogenase activity of
the bifunctional enzyme is responsible for
the oxidative decarboxylation of prephenate
into 4-hydroxyphenylpyruvate in the presence of NAD+ (Figure 2). The aroQt.tyrA
gene, encoding the T-protein from Es. coli,
codes for a 373 amino acid polypeptide with
a molecular mass of 42 kDa.42 aroQt.tyrA
forms a transcriptional unit with aroF and is
part of the TyrR regulon. The divergently
transcribed aroQp.pheA and aroF-aroQt.tyrA
operons are separated by a bifunctional transcriptional terminator. The promoter region
of the aroF-aroQt.tyrA operon contains two
strong and one weak TyrR boxes, which in
the presence of Tyr, bind the TyrR repressor.42,43
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The native AroQt.TyrA enzyme is a dimer
and is retro-inhibited by Tyr (45% for the CM
and 95% for the dehydrogenase activity).44,45
It is now evident that the mutase and the
dehydrogenase active sites are distinct, but
display some similar chemical properties.46-50
Kinetic studies have shown that Tyr binds
twice at distinct allosteric sites to form a less
active Tyr-enzyme-prephenate complex.51 It
has been proposed that the entire TyrA protein family, in contrast to AroQp.PheA, lacks
a discrete allosteric domain, and that inhibitors act competitively at the catalytic site of
different family members which exhibit individuality in the range and extent of molecules
recognized as substrate or inhibitor.52
The mutase activity of the T-protein
(CM-T domain) is very similar to that of the
P-protein, and the N-terminal portions (residues 1–60) of their amino acid sequences are
highly homologous.42 In Er. herbicola, the
aroQt.tyrA gene has been isolated and shows
the same gene organization as in Es. coli.
However, aroQt.tyrA can be expressed efficiently from an internal promoter that appears to lie within the 3′-portion of aroF.
The CM-T domain of the T-protein could be
removed by deletion to form a monofunctional prephenate dehydrogenase.22,53 Similar genes have also been identified in the
genome sequences of Ha. influenzae,32 Pa.
multocida,40 and Vi. cholerae.38
A special case of fused CMs can be found
in the archeon Archaeoglobus fulgidus.54 Here,
a putative trifunctional AroQtp protein has the
CM domain located between a N-terminal
prephenate dehydrogenase domain and a
C-terminal prephenate dehydratase:regulatory
domain.
3. AroQd or Chorismate Mutase
Fused to DAHP Synthase
CM:DAHP synthase is a bifunctional
enzyme of which the N-terminus shows
80
homology with CMs, while the C-terminus
has sequence similarity with DAHP synthases
(or phospho-2-dehydro-3-deoxyheptonate
aldolases). The aroA(G) or aroQd.aroG gene
from Bacillus subtilis, encoding the bifunctional CM:DAHP synthase, has been characterized.55 The corresponding enzyme appears to be a tetramer, containing four
identical subunits of each 358 amino acids.
The DAHP synthase activity is inhibited by
prephenate and chorismate. The bifunctional
enzyme has a single binding site for
chorismate and prephenate that is noncompetitive for the substrates of DAHP synthase.56,57 As a second, monofunctional
DAHP synthase from Ba. subtilis is inhibited by prephenate, it has been postulated
that the bifunctional enzyme arose from the
monofunctional enzyme by the conversion
of the allosteric site used for binding
prephenate into an active site for CM activity.58 In Ba. subtilis the bifunctional polypeptide chain is part of a trifunctional enzyme
complex that also has shikimate kinase activity.59
Only the Ba. subtilis aroQd.aroG gene
has been characterized, and its regulation
has not been examined in great detail. The
expression of the aroQd.aroG gene appears
to be moderately repressed in the presence
of Tyr.60,61 Similar genes encoding putative
AroQd.AroG proteins have been identified
in Ba. halodurans, 62 and Deinococcus
radiodurans.63
4. Nonallosteric, Monofunctional
Chorismate Mutases (AroQf and
AroH)
Monofunctional CMs are involved in
the synthesis of prephenate, which can be
converted to Phe via either phenylpyruvate or arogenate, or to Tyr via either
4-hydroxyphenylpyruvate or arogenate
(Figure 2). In prokaryotic organisms, these
enzymes lack allosteric control by Phe,
Tyr, or Trp. AroQf is found in many Gramnegative bacteria, while AroH is found in
some Gram-positive bacteria, such as Ba.
subtilis64 and Streptomyces aureofaciens.65
In some enteric bacteria, such as Er.
herbicola, AroQp, AroQt, and AroQf coexist.17,53,66
A monofunctional CM encoded by the
aroQf gene was found to be a periplasmic
enzyme in Er. herbicola. The aroQ gene
encodes a 181-residue protein, having a calculated molecular mass of 20.2 kDa. N-terminal amino acid sequencing of the purified
AroQ f protein indicated cleavage of a
20-residue signal peptide. The mature enzyme is a homodimer.66
The smallest natural CM characterized
to date is a thermostable dimer of the AroQf
class and belongs to the hyperthermophilic
archeon Methanococcus jannaschii.13 The
Me. jannaschii aroQf gene codes for a 99
amino acid polypeptide that shows similarity to the CM domain of AroQp proteins
from members of the γ-subdivision of the
proteobacteria. The AroQ protein from Me.
jannaschii shows Michaelis-Menten kinetics and displays pH-independent activity in
the range of pH 5-9.13 Similar putative CM
protein sequences were found in the translated genomes of other archeons, such as
Methanobacterium thermoautotrophicum,67
Aeropyrum pernix,68 and Halobacterium sp.
NRC-1.69
The aroH gene encoding the monofunctional mutase of Ba. subtilis codes for a small
polypeptide of 127 amino acids. The derived
amino acid sequence was found to be marginally similar to a short region of the
N-termini of the two Es. coli bifunctional
P- and T-proteins. The aroH gene product
forms a homotrimer of 14.5-kDa subunits
and is nonallosteric, as its activity is unaffected by any aromatic amino acid.64,70 It
exhibits Michaelis-Menten kinetics, and its
kinetic parameters are similar to those of the
other CMs, suggesting they are functionally
similar. The aroH gene is part of an aroFBH
operon that is organized in a supraoperon
(aroFBH-trpEDCFBA-hisH-tyrA-aroE) for
amino acid biosynthesis. It was shown that
synthesis of AroH is moderately repressed
by the presence of aromatic amino acids.61,71
Similar AroH-encoding sequences have been
identified in Ba. stearothermophilus72 and in
the genome sequences of Ba. halodurans62
and Synechocystis sp. PCC6803.73
5. AroQr or Allosteric,
Monofunctional Chorismate
Mutases
These monofunctional CMs are characteristic for eukaryotic microorganisms, such
as Saccharomyces cerevisiae, and plants.
They exhibit allosteric inhibition by Phe and/
or Tyr and allosteric activation by Trp. The
ARO7 gene from Sac. cerevisiae codes for a
256 amino acid polypeptide of 29.7 kDa. In
contrast to some other genes involved in
aromatic amino acid synthesis, the ARO7
gene is not regulated at the transcriptional
level. It is not derepressed by the general
amino acid control system, as no consensus
for the GCN4 activator protein binding site
was found.74 The enzyme is a dimer and its
activity is under dual control as it is modulated by a tenfold activation by Trp and a
tenfold inhibition by Tyr. The allosteric regulation site is distinct from the active site.74,75
The enzyme shows positive cooperativity
toward the substrate, which is lost after the
addition of Trp.75 The different conformations of an allosteric enzyme can be described to consists of an equilibrium between
an activated R- (relaxed) state and a repressed
T- (tense) state. The R-state of the yeast CM
has a pH optimum of 7, while the T-state
shows a pH optimum of 5.75 A Thr226Ile
mutant shows a tenfold-increased basal level
enzyme activity and is unresponsive to Tyr
81
and Trp regulation.74 The mutant enzyme
appears to be locked in the activated R-state.75
In the methylotrophic yeast Hansenula
polymorpha, the HARO7 gene codes for a
CM that in terms of catalytic and structural
properties is very similar to the Sac.
cerevisiae CM. However, HARO7 gene expression is induced in the presence of methanol, suggesting catabolite repression.76 In
Aspergillus nidulans a highly similar, monofunctional allosterically regulated CM was
characterized. The encoding AROC gene
codes for a 268 amino acid polypeptide, and
its expression is not regulated at the transcriptional level.77
C. Crystal Structures, Active Sites,
and Reaction Mechanisms
The recent determination of the crystal
structure of three CMs has led to a better
understanding of the reaction mechanism,
which is already under investigation for at
least 3 decades. The reaction is a pericyclic
process, a Claisen rearrangement, which involves the cleavage of the C5-O7 ether oxygen bond followed by C1-C9 bond formation (Figure 3). The chorismate to prephenate
rearrangement is strongly exergonic and essentially has an irreversible nature.77 In solution, the flexible chorismate molecule preferentially adopts the pseudoequatorial
conformation in dynamic equilibrium with
the higher-energy pseudodiaxal form.78 The
uncatalyzed rearrangement occurs readily in
aqueous solution and seems to be a concerted asynchronous reaction that proceeds
via a transition state with chair-like geometry.79,80 Molecular orbital calculations, substrate labeling, and kinetic isotope effect studies demonstrated that both the uncatalyzed
and catalyzed reactions proceed through a
chair-like transition state in which the C5O7 bond cleavage precedes C1-C9 bond
formation, resulting in a dipolar transition
82
state.78-83 The CM enzyme catalyzes this reaction with a 2 × 106-fold rate enhancement
over the uncatalyzed process.84 For the study
of the reaction mechanism of CM, an endooxabicyclic transition state analog, which is
a competitive inhibitor for prephenate, was
used frequently (Figure 3).85 CM might accelerate the conversion of chorismate to
prephenate by various mechanisms, including carbocation formation at C4, protonation
of the ether oxygen, nucleotrophic attack at
C5, or stabilization of the putative dipolar
transition state for the concerted pericyclic
reaction.78
1. Monofunctional Chorismate
Mutase of Bacillus subtilis
The first CM protein structure determined
was that of the monofunctional CM of Ba.
subtilis, belonging to the AroH class
(1com.pdb, 2chs.pdb, 2cht.pdb).86,87 It was
shown that a monomer folds into a fivestranded mixed β-sheet, a 19-residue α-helix, and two 310-helices. The trimer structure
could be described as a pseudo-αβ-barrel
with the β-sheets from each monomer forming the core and helices on the outside. The
C-terminal 12 residues are disordered in the
crystals of the unliganded protein.86,87 The
resolution of a CM complex with the transition state analog of prephenate, revealed that
the three equivalent active sites are located
at the interface of two adjacent subunits. No
functional group capable of proton transfer
to the ether oxygen could be found at the
active site.87 Moreover, the rate-determining
transition state of the Ba. subtilis monofunctional CM (BsCM) is insensitive to solvent
deuteration and to large variations in the pH
of the solution.88 These observations argue
against the involvement of a catalytic residue, suggesting that the rearrangement itself
appears to be rapid via a single pericyclic
process, similar to the uncatalyzed reaction,
FIGURE 3. The Claisen rearrangement of chorismate to prephenate. The reaction occurs via a chair-like
transition state, which is mimicked by the transition state analog, endo-oxabicyclic acid. (This figure was
adapted from Walsh et al.11)
as was confirmed by 13C-NMR and FTIR
studies.70,89
Numerous interactions of hydrophobic,
ionic, and polar nature between the enzyme
and the inhibitor are present (Figure 4A). This
suggests that chorismate itself is bound to the
enzyme in a pseudodiaxal chair-like conformation with its enolpyruvyl side chain positioned over the cyclohexadiene ring.86 Arg7,
together with Arg90, form hydrogen bonds to
the C11 carboxylate group of chorismate and
are proposed to play an important role in positioning the enolpyruvyl side chain in the chairlike transition state.87,90 Moreover, it has been
shown that the bound conformer of chorismate
has a distorted geometry, reducing the C1-C9
distance between the reaction centers.91 Specific electrostatic interactions probably stabilize the presumed dipolar state in the BsCM
catalyzed rearrangement.91,92 The developing
positive charge on the cyclohexadienyl ring
may be stabilized by the anionic carboxylate of
Glu78, while the developing negative charge
of the ether oxygen could be accommodated
by electrostatic interactions with the positively
charged guanidinium group of Arg90.86,93 Indeed, the positive charge of Arg90 and the
negative charge of Glu78 have been shown to
be essential for the catalytic activity of the
enzyme.90,94,95 This has been supported by further crystallographic analysis of two CM double
mutants (1fnj.pdf, 1fnk.pdf).96
The binding of ligand induces structural
alterations in the C-terminal segment of the
protein, which becomes highly ordered, due
to interactions between the side chain of the
substrate and Leu115. As this C-terminal
segment is situated immediately adjacent to
the active site, its ordering may cap the active site after substrate binding and affect
efficient catalysis and likely influences the
association and dissociation rates of the enzyme and its ligands.86,89 Indeed, the reaction appears to be limited by substrate binding and product release at low and high
substrate concentrations, respectively, and is
partially diffusion controlled.88,97 Recently,
a high-resolution BsCM crystal structure
83
FIGURE 4. Active sites of the CMs from (A) Ba. subtilis, (B) Es. coli, and (C) yeast. The hydrogen bonds and
electrostatic interactions with the transition state analog are shown. The active site of the allosteric CM from
yeast was proposed based on a superposition with the Es. coli active site. (The figure was adapted from
Schnappauf et al.,112 Cload et al.,90 and Liu et al.103
(1dbf.pdb) supports the flexibility of the Cterminal tail, which could make it possible
for the C-terminal tail to actually participate
in the reaction mechanism.98 However, mutational analysis of the C-terminus suggests
it does not participate directly in the chemical arrangement, but appears to contribute to
enzymatic efficiency via uniform binding of
the substrate and transition state.99
2. The ‘Mini-Mutase’ of Escherichia
coli
The N-terminal 109 residues of the
Es. coli P-protein constitute a fully functional CM domain. The crystal structure
(1ecm.pdf) of this monofunctional ‘mini-
84
mutase’ (EcCM) shows that the engineered
CM forms a dimer.100 Each monomer consists of three α-helix domains separated by
two short spacer regions. The structure of
the EcCM, complexed with the transition
state analog, identified two equivalent active
sites in the dimer, each with contributions
from both monomers. The transition state
analog is completely buried within the active site, in contrast to the BsCM, where the
analog is partially solvent exposed. Although
the secondary structure and folds of the
BsCM and the EcCM, respectively, are completely different, their active sites are highly
similar (Figure 4B). Arg11 in EcCM, as Arg7
from BsCM, interacts with the C11 carboxylate, while the C4-hydroxyl group interacts
with Glu52 in EcCM and Glu78 in BsCM.
Lys39 of EcCM that interacts with the C11
carboxylate and the ether oxygen O7 has an
equivalent position and identical charge with
the Arg90 residue of BsCM. These residues,
which share the highest degree of similarity,
create a highly charged active site. Compared with BsCM, there is in EcCM an additional interaction between the ether oxygen and the active site, in the form of a
hydrogen bond with Gln88.100
Lys39 (Lys37 in the T-protein) is conserved among several CMs and was shown
to be essential for mutase activity in the
T-protein from Es. coli.46 Based on the variations with pH of the CM-T kinetic parameters, a possible role for Lys37 as a catalytic
general acid has been proposed.46,101,102 However, mutational analysis of Arg11, Glu52,
Gln88, and Lys39 from EcCM confirmed a
major role of these residues in orientational
positioning and/or catalysis, but argued
against a general acid/base catalysis.103 Accordingly, it has been proposed that Lys39
from the EcCM, aided by the Arg11, Arg28,
and Arg51 groups, is involved in the conformational trapping of chorismate, by rearranging the chorismate side chain to a
pseudodiaxal conformation.100,104
Wild-type Es. coli CM (P-protein)
shows a constant activity at a broad pH
range. At pH > 7.5, a Gln88Glu mutation
results in an inactive enzyme. 103 To maintain the same total charge at the active
site and the hydrogen bond to the ether
oxygen, the carboxylate group of Glu88
is inferred to be protonated in the active
enzyme, and this substitution at the active site has been proposed to account for
the different pH profiles of the Es. coli Pprotein (Gln88), the Es. coli T-protein
(Glu86) and yeast CM (Glu246, see below). Indeed, it was shown that the charge
status of residue Glu88 in the mutant plays
an important role in the catalytic process
but has only a small impact on inhibitor
binding.103,105,106
3. The Allosteric Chorismate Mutase
of Saccharomyces cerevisiae
The first member of the AroQr group for
which the crystal structure was determined is
the Thr226Ile mutant from Sac. cerevisiae.
Subsequently, the crystal structure of the Tyrbound T-state and the Trp-bound R-state of
wild-type CM were determined (1csm.pdf,
2csm.pdf, 3csm.pdf, 4 csm.pdf, 5 csm.pdf).107111 As a relatively small protein of the allosteric family, yeast CM (YCM) provides an
ideal model system for exploring the detailed
mechanisms of allosteric regulation. The
monomer structure consists of 12 helices,
connected by loops, that form a twisted twolayer structure. The overall shape of the dimer
is a bipyramid, with four helices (H2, H4, H8,
and H11) involved in dimerization.108
By comparison with EcCM, the active
site of YCM was determined (Figure 4C).
Both active sites are embedded within similar
four-helix bundle units, although with different topology. Four active site residues are
conserved: Arg16 (Arg11 in EcCM) an
Arg157 (Arg28), which bind the inhibitors
two carboxylates, Lys168 (Lys39), which
binds the ether oxygen and Glu198 (Glu52)
which binds the inhibitors hydroxyl group.106
Both YCM and EcCM possess a pronounced
dipolar active site with Glu198 (Glu52), and
Lys168 (Lys39) providing the negative and
positive charge, respectively, and it is likely
that these residues fulfill roles similar to Glu78
and Arg90 in BsCM.106,110 Mutational analysis of all critical active site residues in YCM
confirmed the proposed location of the active
site and quantified their contribution to catalysis.112 Glu246 (cf. the EcCM Gln88Glu
mutant and T-protein Glu86) in the catalytic
center forms a hydrogen bond, either directly
or via a water bridge, to the ether oxygen and
must be protonated for maximum catalysis. It
has an increased pKa value resulting in retention of a proton on Glu246 to a higher pH.
However, it restricts optimal activity of the
85
enzyme to low pH.106,109,110,112,113 It may function as a polarizing group for the ether-oxygen of chorismate but could also be involved
in protonating the ether-oxygen.112 However,
the inhibitor binding mode supports a mechanism by which polar side chains of the enzyme bind the substrate in the pseudodiaxal
conformation, and no evidence was found to
support the involvement of specific catalytic
groups.109,110
Both Trp and Tyr bind the same hydrophilic channel through the center of the dimer,
indicating that there is only one regulatory
binding site per monomer. The allosteric
binding site involves helix H8 of one monomer and helices H4 and H5 and the L80s
loop of the other monomer. Both effectors
bind to common residues of the longest helix
(H8), which spans the whole monomer and
extends from the regulatory site to the active
site.108,110,114 The six phenyl atoms of Trp
show strong van der Waals interactions with
the protein, and the steric side chain of Trp
pushes apart the allosteric domain of one
monomer and helix H8 from the catalytic
domain of the other monomer.108 The phenolic hydroxyl group and the carboxylate of
Tyr interacts specifically with Thr145 and
the L80s loop, thereby bringing the allosteric and catalytic domains together. So,
dimer formation is essential for effector binding and allosteric regulation. The allosteric
transition from T to R can be seen as a 15°
rotation of the catalytic domains of the dimer
relative to each other.111 In the presence of
the transition state analog a super R-state is
formed, the monomer-monomer angle of
which differs by 22° from the T-state.110
The rotation of the two catalytic domains
relative to each other, associated with the
binding of Trp, also involves the movement
of the L220s loop between two helices, H11
and H12, both of which contribute to the
active site. As a result many interactions at
the dimer interface (H2 and H11) are rearranged and cause further movement within
86
the catalytic domain.110,111,115 Binding of Tyr
to the allosteric site causes structural changes,
which include alternative conformations
around Tyr234 in H12. Tyr234 interacts via
Glu23 with the catalytic site residue Arg157,
which probably strongly affects substrate
binding.110,115,116 Changes in the adjacent
L220s loop can affect this molecular trigger
and ultimately result in enzymes that are
unresponsive to Tyr.117 Thr226 (from the
Thr226Ile mutant) is the last residue of the
L220s loop next to helix H12 and probably
influences the conformation of this loop. A
double mutant Thr226Ile/Ile225Thr unlocks
the R-state and restores activation by Trp but
not the inhibition by Tyr. This is consistent
with an intermediate structure between the
T- and R-states, corresponding with the
unliganded enzyme. It was suggested that
the molecular trigger of the T-state induced
by Tyr and activation of the enzyme by Trp
are independent processes and use alternative pathways within the molecule.114,115 The
Thr226 residue is not conserved in the CMs
from Ha. polymorpha and As. nidulans.
Mutations of the corresponding Asp residue
in As. nidulans indicate that, despite the high
similarity with YCM, the regulation of the
enzyme activity uses different intramolecular signal transduction pathways.76,77
4. Conserved Reaction Mechanism
An unusually wide variety of primary
structures can serve as effective catalysts for
the mutase transformation. This view is supported by the isolation of monoclonal antibodies raised against the transition state analog of CM, with impressively high catalytic
potency for the mutase reaction.118,119 The
crystal structure of one of these catalytic
antibodies (1F7) has been determined
(1fig.pdb) and suggests that the antibody
stabilizes the same conformationally restricted pericyclic transition state.93 More-
over, there is an overall similarity between
the catalytic mechanisms employed by the
antibody and CM enzymes. By the use of
directed evolution, the enzyme topology of
the dimeric CM of Me. jannaschii was redesigned to yield an active, monomeric fourhelix-bundle protein.120-122
There are two fundamentally different
architectures for CM enzymes, but their active sites are similarly functionalized, exhibiting comparable kinetic parameters. The
active sites exploit an extensive and analogous set of hydrogen bonding and electrostatic interactions to bind the ligands.97 CM
is a classic example of induced fit and may
well be one of the simplest of enzymes in
mechanistic terms.89 However, the exact
mechanism of the enzyme-catalyzed rearrangement remains uncertain. In conclusion,
it can be stated that the CM enzyme restricts
the conformational degrees of freedom of
the flexible chorismate molecule and procures the selection of the less stable but reactive pseudodiaxal conformer.123,124 The
enzyme creates a strong hydrophobic environment, and the electrostatic gradient in the
active site is a major factor in catalysis, as it
stabilizes the dipolar transition state.89,95,113,125
All these factors together probably lead to a
million-fold enhancement of the reaction.86,89,91,93-95,113
aromatic acids.1 The corresponding cDNA
of CM-1 of Ar. thaliana encodes a protein of
334 amino acids, which shows 41% amino
acid identity with YCM and can be denoted
as an AroQr CM. The N-terminal portion of
the protein resembles known plastid-specific
transit peptides.128 The cDNA of the cytosolic, unregulated CM (CM-2) of Arabidopsis
thaliana codes for a 265 amino acid polypeptide, which lacks the N-terminal transit peptide. It shows 50% amino acid identity to
CM-1.129 A third CM (CM-3) is also allosterically regulated and has a putative plastid
transit peptide but exhibits a significantly
higher apparent KM value for chorismate than
CM-1.127 In tomato, the cDNA encoding a
cytosolic CM-2 type CM of 255 amino acids
has been isolated.130
No CMs have been found in animals,
with some exceptions. The esophageal gland
of a root-knot nematode Meloidogyne javanica
produces a potentially secreted CM, which
resembles bacterial CMs. The nematode CM
is not retro-inhibited by aromatic acids and
could be assigned to the AroQf subgroup. It
may reflect past entry of a bacterial CM gene
into the nematode genome by horizontal gene
transfer.131 A similar secreted CM has also
been identified in the potato cyst nematode
Globodera rostochiensis.132
E. Chorismate Mutase Homologs
D. Chorismate Mutases in Other
Organisms
In plants, both Tyr and Phe are synthesized via prephenate and arogenate. CM is
monofunctional and exists in often two (CM1 and CM-2) or even three (CM-3) separable
isoenzymes, which have distinct physiological roles in coordinating CM activity with
developmental and environmental signals.126,127 CM-1 is activated by Trp and retroinhibited by Phe and Tyr, whereas the second isoenzyme CM-2 is insensitive to the
The antibiotics candicidin, chloramphenicol and pristanimicin I produced by Streptomyces griseus,133 St. venezuelae,134 and St.
pristinaespiralis,135 respectively, were shown
to have a p-aminophenylalanine precursor. The
p-aminophenylalanine precursor is derived from
chorismate via 4-amino-4-deoxychorismate
(ADC).136 An ADC mutase subsequently converts ADC to 4-amino-4-deoxyprephenate, leading to p-aminophenylanaline.11 Such an ADC
mutase was identified in St. pristinaespiralis
and is encoded by papB. The deduced amino
87
acid sequence shows marginal homology to
AroQ CMs. Both enzymes catalyze a similar
reaction, in that their substrates differ only in the
para-position.
III. ANTHRANILATE SYNTHASE
A. Introduction
The biosynthesis of Trp from chorismate
involves five enzymatic reactions and is
encoded by seven trp genes.137 Anthranilate
synthase (AS; EC 4.1.3.27) catalyzes the first
committed step in Trp biosynthesis in which
chorismate is aminated and then aromatized
to anthranilate (o-aminobenzoate), concomitant with the loss of the enolpyruvylgroup as
pyruvate. It creates a key biosynthetic branch
point, feeding anthranilate down the sole
pathway in nature capable of generating a
heterocyclic indole ring from nonaromatic
precursors. The enzyme generally consists
of two nonidentical subunits, α and β, which
associate to give αβ dimeric or α2β2 tetrameric heteromers.3 The AS α-subunit can
by itself act as an AS when NH3 is present.138
AS subunit β belongs to the triad class, or
TrpG type, glutamine amido transferases
(GATases) and facilitates anthranilate synthesis by transferring the amide function from
Gln to the α-subunit.139 As AS determines
the flux of precursor into the Trp biosynthetic pathway, its expression is highly regulated. Moreover, AS is subject to retro-inhibition by the end product of the pathway,
Trp. For a review on the enzymology of AS
in microorganisms and plants, the reader is
directed to Romero et al.3
B. Gene Organization and
Regulation
The organization and regulation of trp
genes have been studied extensively for a
88
great variety of organisms and varies considerably between species.140 The AS α- and
β-subunits are encoded by the trpE and trpG
genes, respectively, in prokaryotic organisms. However, the β-subunit can sometimes
be part of a multifunctional enzyme encoded
by other trp genes. An impressive number of
AS-encoding genes have been isolated and
characterized from diverse bacterial and fungal species. In most cases, the expression of
the AS genes is highly regulated by diverse
mechanisms such as negative regulation by
repression, attenuation effected by translation, attenuation effected by RNA-binding
proteins, and attenuation effected by tRNA
binding. As it is beyond the scope of this
review to discuss all reported AS genes and
their regulation, we focus on a number of
model organisms that display the different
organizations and regulations that can be
found.
1. Escherichia coli
In Es. coli and Sal. typhimurium, all the
trp genes are organized in a trpEG(D)C(F)BA
operon.141 The AS β-subunit is N-terminally
fused to phosphoribosyl-anthranilate transferase, which catalyzes the subsequent step
in Trp biosynthesis, and is encoded by the
trpG(D) gene.142 The start and stop codon of
trpG(D) and trpE overlap, respectively, indicating translational coupling of these genes
that facilitates the equimolar synthesis of the
polypeptides for the enzyme complex.143 The
operon is under dual transcriptional control
exerting repression and attenuation of transcription. Repression is the primary mechanism of control for Trp biosynthesis. Attenuation contributes significantly to control,
but only under conditions of Trp starvation,
when repression is relieved.144,145
Repression of the trp operon is mediated
by the Trp-activated TrpR repressor in function of the intracellular concentration of Trp.
The trpR gene codes for a 108 amino acid
polypeptide.146 TrpR aporepressor dimers
bind two molecules of the co-repressor
ligand, Trp, to form a holorepressor complex with increased operator affinity.147,148
The helix-turn-helix DNA-binding motif of
the Trp repressor (1wrp.pdb, 2wrp.pdb,
3wrp.pdb) recognizes a symmetric 18-bp site
in the trp operator, which contains two palindromic CTAG elements separated by a
spacer of 4 bp.149-153 The operator of the trp
operon overlaps the -10 region of the trp
promoter, and the binding of the repressor
excludes the binding of RNA polymerase.
The TrpR protein is a global regulator of
gene expression and binds sites in at least
five different operons, all related to Trp biosynthesis and transport.154,155
Upstream of trpE, a leader region was
found, which is responsible for attenuation
control by Trp. The leader sequence consists
of a 14 amino acid short leader peptide coding sequence, trpL, which contains two consecutive Trp codons. The mRNA in the leader
region can form mutually exclusive stemloop structures, including a pause, terminator, and antiterminator structure. Transcription termination at the terminator is regulated
by the concentration of charged tRNATrp,
mediated via the translation of trpL. The
working mechanism of attenuation control
of the trp operon has been investigated in
detail by the use of different mutations in the
leader region.156-160
A similar organization and regulation is
found in all investigated enteric bacteria.161
For example, the trp genes from Serratia
marcescens are organized in a single operon.
The upstream region displays a very similar
promoter/operator/leader region as the Es. coli
trp operon.162,163 However, the trpG gene of Se.
marcescens is not fused to the adjacent trpD
gene and suggests gene fusion events during
the evolution of the trp operon in Enterobacteriaceae.142,164 Dual repression and attenuation
control of the trpEGDC(F)BA operon has also
been found in the Gram-positive bacterium
Brevibacterium lactofermentum.165-167 A putative leader region involved in attenuation is
present upstream the trpEG(D)C(F)BA operon
of Corynebacterium glutamicum168 and the
trpEG cluster of Thermus thermophilus.169
2. Bacillus subtilis
In Ba. subtilis six of the seven trp genes
are organized in a trpEDCFBA operon, which
is part of a His and aromatic amino acid
biosynthetic supra-operon.170 The trpG gene
codes for an amphibolic GATase, which is
also involved in p-aminobenzoate biosynthesis (see Section IV.B). Hence, the trpG
gene is located in a folate biosynthetic operon.171 The trpEDCFBA genes are transcribed from a constitutive promoter. A trp
leader transcript can form mutually exclusive secondary structures: an antiterminator
and a rho-independent terminator.172 The
expression of the trp biosynthetic genes in
Ba. subtilis is regulated by the trp RNAbinding attenuation protein (TRAP).173 The
TRAP complex contains 11 identical subunits of 75 amino acids each, encoded by
mtrB.174-177 They are arranged in a doughnutlike structure (1wap.pdb, 1c9s.pdb) termed
the β-wheel.175,178,179 Each subunit contains
three basic amino acids that form a KKR
motif (Lys 37, Lys56, Arg58) that constitutes the RNA-recognition domain of
TRAP.178,180 The TRAP complex is activated
by Trp, which binds in a highly cooperative
manner at a site between adjacent subunits.181,182 The trpEDCFBA operon is regulated by an attenuation mechanism in which
Trp-activated TRAP binds with its KKR
motif in each subunit to 11 (G/U)AG repeats
separated by 2- to 3-nucleotide spacers in
the trp leader transcript, six of which are
present in the antiterminator structure.183-186
TRAP binding blocks the formation of the
antiterminator structure and thereby promotes
89
transcription termination at the terminator.
A 5′-upstream RNA stem-loop structure also
interacts with TRAP and probably increases
the likelihood that TRAP will bind to the (G/
U)AG repeats in time to block antiterminator
formation.187
Upstream of the trpE Shine-Dalgarno
sequence, a secondary RNA structure can be
formed. The binding of TRAP in this region,
which contains 11 (G/U)AG repeats, promotes refolding of the RNA such that the
trpE Shine-Dalgarno sequence is sequestered
in a stable RNA hairpin, thus inhibiting TrpE
synthesis.188 TRAP also regulates the translation of trpG in the folate operon by binding nine (G/U)AG repeats that overlap the
ribosome binding site, thereby blocking translation.189,190 A mutant in trpS, encoding
tryptophanyl-tRNA synthase, showed a
higher expression of the trp genes. This
implicates a role of tRNATrp in the activation
of TRAP.191 Consistently, an operon of Ba.
subtilis was identified that responds to uncharged tRNATrp by producing a transcript
that limits the action of TRAP.192 Recently,
the yhaG gene of Ba. subtilis, involved in
Trp transport, was also found to be regulated
by TRAP.192,193 A similar TRAP-regulated
attenuation mechanism is also present in Ba.
pumilis,194-196 and Ba. stearothermophilus
(1qaw.pdf).72
3. Lactococcus lactis
Yet another form of attenuation control
can be found for the trpEGDCFBA operon of
Lactococcus lactis in which the binding of a
deacylated tRNA to a nontranslated leader
regulates transcription termination.197-199 The
La. lactis trp leader shows structural similarity to the leader transcripts of a group of
Gram-positive aminoacyl-tRNA synthase
genes and some amino acid biosynthetic operons that are controlled by antitermination
through interaction of the leader transcript
90
with cognate uncharged tRNA. These leader
RNAs each contain three conserved stemloop structures followed by a highly conserved 14-base element (T-box) and the attenuator-specified terminator hairpin. A model
was proposed in which antitermination is
mediated by stabilization of the antiterminator
conformation of the leader mRNA through
interaction of the T-box with uncharged
tRNATrp.
4. Sinorhizobium meliloti
In Si. meliloti, a single gene encodes both
the AS α-subunit and β-subunit activities.
This trpE(G) gene fusion codes for a 729
amino acid polypeptide in which the C-terminus of the α-subunit joins the N-terminus of
the β-subunit through a short connection segment.200 The gene is preceded by a leader
region which can form putative secondary
mRNA structures and contains a coding region for a short leader peptide, TrpL, with
three consecutive Trp residues.200 Attenuation control as in Es. coli has been shown.201,202
The transcription start has been determined
and coincides with the first base pair of the
start codon of the trpL leader sequence. This
implies that there is no ribosome binding site
for the trpL sequence. A similar gene fusion
has also been found in the translated genome
sequence of Mesorhizobium loti203 and has
been isolated from Azospirillum brasilense204
and St. venezuelae.205 In these cases, a trp
attenuator region, encoding a putative leader
peptide containing three consecutive Trp residues, is present upstream of the trpE(G) gene
fusion. A fused AS has also been detected in
the eukaryote Euglena gracilis.206
5. Pseudomonads
In Ps. aeruginosa and Ps. putida the trpE
gene is solitary, while trpG is the first gene in
a three-gene operon containing trpD and
trpC.207,208 In Ps. putida, trpE is 2.2 kb upstream from the trpGDC cluster, whereas in
Ps. aeruginosa they are separated by at least 25
kb, which contains a large cluster of pyocin R2
genes.209 In both organisms mutants have been
isolated that constitutively and simultaneously
overproduce TrpE, TrpG, TrpD, and TrpC,
suggesting the presence of a common regulatory mechanism, formally analogous to the
TrpR-mediated repression in Es. coli. In wildtype Ps. aeruginosa and Ps. putida, however,
Trp did not alter the expression of the same
genes, which indicates that they are always
fully repressed by the intracellular concentration of Trp.210,211 Similarities in the 5′untranslated regions of trpE and trpGDC in
both pseudomonads support the presence of
common regulatory elements.207,208 Expression
analysis of the trpE gene from Ps. syringae
subsp. savastanoi showed that its transcription
was independent of the Trp concentration.212
Similar results were obtained with Caulobacter
crescentus and Chromobacterium violaceum,
where the trp genes appear not to be regulated
at the transcriptional level.213,214
6. Buchnera spp.
A special mechanism for increased Trp
biosynthesis has been found in the aphid endosymbiont Buchnera spp. The trpE and trpG
genes have been amplified and reside as one
or more trpEG repeats on a plasmid, while the
trpDC(F)BA genes are located on the chromosome. The transcription of the genes is
constitutive, leading to increased AS activity
and hence Trp production. In some Buchnera
lineages a silencing of these trpEG copies
resulted in trpEG pseudogenes.215-221
7. Saccharomyces cerevisiae
In yeast, AS subunit α is encoded by
TRP2, which is located on chromosome V
(Ref. 222). AS subunit β is fused to indole3-glycerol phosphate synthase, which catalyzes the penultimate step in Trp biosynthesis.223 The fused protein is encoded by
the TRP3 gene, which is located on chromosome XI.224 Both genes are part of a
complex regulatory network known as general amino acid control. The regulator protein GCN4 activates transcription in the
general control system.225 GCN4 belongs to
the basic region leucine zipper family of
DNA-binding proteins, as it contains a bipartite DNA-binding motif consisting of a
leucine zipper dimerization domain and a
highly charged basic region that directly
contacts DNA. The transcription factor
forms a dimeric complex, with each monomer recognizing half of a symmetric or
nearly symmetric DNA-binding site, known
as a GCN4 recognition element.226 Both
TRP2 and TRP3 are regulated coordinately
as, in both genes, a GCN4 consensus sequence with a single mismatch is located in
the promoters.227 It was shown that the
GCN4 activation of TRP3 is enhanced by
the general regulatory factor ABF1.228 In
some fungi, the AS β-subunit is part of a
trifunctional GATase/indole-3-glycerol
phosphate synthase/phosphoribosyl-anthranilate isomerase enzyme, which is encoded
by the TRP1 gene in Neurospora crassa229
and Flammulina velutipes230 and the TRPC
gene in As. niger.231
C. Active Site Residues and
Reaction Mechanism
Many amino acid sequences have
been published for both the α- and
β-subunits of the AS enzyme. The alignment of the sequences for the α-subunit
of different microorganisms shows that
the sequence can be divided into an
N-terminal region of low sequence conservation and a highly conserved C-terminal region.
91
1. Allosteric Regulation
Almost all identified ASs are retro-inhibited by Trp.3 Different retro-inhibition-resistant AS mutants have been isolated, by selecting for 5-methyl-Trp or 5-fluoro-Trp resistance.
Analysis of such mutants in Sal. typhimurium,232
Br. lactofermentum,167 and Sac. cerevisiae233
indicated two amino acid clusters, LLESX10S
and NPSPYM, to be essential for the allosteric
regulation by Trp. Both clusters are highly
conserved throughout the different AS sequences and are located in the N-terminal region. Both clusters are thought to interact to
constitute the Trp-binding site,233 as confirmed
by crystal structure analysis (see Section
III.C.4.) Both retro-inhibition motifs can be
recognized in the amino acid TrpE fusion sequences of Si. meliloti and Az. brasilense.
However, some crucial amino acids are not
conserved. The archeon Thermococcus
kodakaraensis Trp protein even lacks the
LLESX10S cluster. Nevertheless, retro-inhibition by Trp has been shown in Si. meliloti and
and in The kodakaraensis.202,205 A hybrid AS
tetrameric complex containing one catalytically active, retro-inhibition-insensitive and one
catalytically inactive, retro-inhibition-sensitive
mutant α-subunit from Sal. typhimurium, demonstrated that the binding of a single Trp inhibitor molecule to one α-subunit is sufficient
for the propagation of a conformational change
that effects the active site of the other α-subunit.234
2. Anthranilate Synthase Activity
In the C-terminal region of AS, some
highly conserved residues were shown to be
involved in catalysis. In Se. marcescens, an
Arg, a His, and a Cys residue were identified
as essential by chemical modification experiments.235 Mutagenesis of the corresponding Arg residue and His residue in
Ba. caldotenax and Sal. typhimurium,
92
respectively, confirmed their role in AS activity.236,237 Additional residues in the C-terminal part of the Sal. typhimurium TrpE sequence were shown to be critical for catalytic
activity.238 These residues are among those
that are highly conserved or invariant.
AS catalyzes the formation of anthranilate,
Glu, and pyruvate from the precursors
chorismate and Gln (Figure 5). trans-6-amino5-[(1-carboxyethenyl)-oxy]-1,3-cyclohexadiene-1-carboxylate, commonly called
aminodeoxyisochorismate (ADIC), was
shown to be an intermediate in the enzyme
reaction.239-241 A His398Met mutant of TrpE
from Sal. typhimurium accumulates ADIC
and indicates that AS subunit α is in fact a
bifunctional enzyme converting chorismate
to anthranilate in two discrete steps.237 The
first step involves the reversible amination of
chorismate to ADIC by ADIC synthase in
the presence of Mg 2+. In analogy with
isochorismate synthase (see Section V.C) and
by using specifically designed inhibitors, it
was shown that the reaction probably involves
a direct substitution of the C4 hydroxyl group
with the C6 ammonium group via a concerted
syn-1,5 displacement of hydroxide by ammonium. This reaction scheme involves a Mg2+bound transition state, in which Mg2+ chelates
the 4-OH group, making it a better leaving
group, and helps to deliver the incoming ammonium group. This would require the binding of the pseudodiaxal conformer of
chorismate.11,242,243 ADIC remains enzyme
bound and ADIC pyruvate-lyase catalyzes
in a second irreversible step the cis-elimination of pyruvate and the aromatization
to anthranilate. This step probably involves
the abstraction of the proton at C6 of ADIC
or protonation of the enol-pyruvyl moiety,
which might point to the possible role for
the conserved His398 residue. 11,244 The
ADIC synthase activity can be functionally uncoupled from the ADIC pyruvatelyase activity and both activities are sensitive to retro-inhibition by Trp.237
FIGURE 5. The formation of anthranilate from chorismate by the bifunctional AS. The ADIC synthase reaction
occurs via a concerted 1,5 double SN2’ displacement mechanism. The transition state involves a C4-C5-C6-N-MgO chairlike six-membered chelate ring system. ADIC pyruvate-lyase catalyzes the cis-elimination of pyruvate and
the subsequent aromatization to anthranilate. (This figure was adapted from Walsh et al.11 and Kozlowski et al.242)
3. Glutamine Amidotransferase
Activity
The AS β-subunit belongs to the triad subfamily of GATases, in which three regions are
highly conserved. One region contains the
unique, invariant Cys residue, another contains
conserved His and Glu residues, which might
act as general acid/base catalysts.244 The three
residues are postulated to form a catalytic
triad.139,245 In both Se. marcescens and Ps.
putida, the role of the Cys residue (Cys83 and
Cys79, respectively) was established.246-249 Replacement of the conserved His in Es. coli
(His170) with Tyr resulted in a complete loss
of GATase activity. This His170 is postulated
to act as a general base that functions to generate the cysteinyl nucleophile needed to attack the carboxamide of Gln.250 In addition, a
conserved Lys residue associated with the active site was modified in Se. marcescens, resulting in a reduced reactivity of the essential
Cys residue.251 The β-subunit probably hydrolyzes the co-substrate Gln via a γ-glutamyl-S-
cysteinyl enzyme intermediate to release ammonium for the amination reaction.250 A protonated form of the conserved His or Glu may
participate in amide release. The nascent ammonium, which is formed at the GATase active site, is unable to equilibrate with the bulk
solvent and is channeled to the active site on
subunit α for the amination reaction. The inactivation of the Gln-dependent AS activity by
the use of Gln analogs depends on the presence
of the substrate chorismate.246 This indicates
that chorismate binding of the α-subunit precedes the binding of Gln to the β-subunit and
promotes a conformational change that is essential for the formation of the subunit βglutaminyl covalent intermediate and for hydrolysis of Gln.252
4. Crystal Structure of Anthranilate
Synthase
The crystal structure of unliganded AS
(1qdl.pdb) from the hyperthermophile archeon
93
Sulfolobus solfataricus was the first to be
determined.253 AS of Su. solfataricus is a
heterotetramer, in which two TrpG-TrpE
protomers associate mainly via the TrpG subunit.253,254 The TrpE subunit of 421 residues has
a complicated α/β-folding pattern of novel topology with two domains and a cleft. Domain I,
which consists of a nine-stranded antiparallel
β-sheet and four helices, forms an orthogonal
β-sandwich with domain II, which consists of a
nine-stranded antiparallel β-sheet and six helices. The conserved residues that were shown to
be important for catalysis are located on two
internal surfaces of the hydrophobic cleft. The
two clusters, shown to be involved in retroinhibition by Trp, are found in domain I, clustered on one side of the orthogonal β-sandwich.253
However, the complicated fold of the α-subunit
rules out the proposed distinction between a
N-terminal regulatory and a C-terminal catalytic domain.232,255
The β-subunit of 195 residues has the known
triad GATase fold. The core of the compact,
spherical α/β-structure is an open, sevenstranded, mixed β-sheet, which resembles the
known structures of other members of the triad
GATases, like guanosine monophosphate synthase and carbamoyl phosphate synthase. The
residues of the catalytic triad (Cys84, His175,
Glu171) are at identical positions as in the abovementioned GATases, ready for catalysis of the
glutaminase reaction. The tips of the α-subunit
sandwich contact the β-subunit across its active
site triad. The active site residues of the
α-subunit and some of the retro-inhibition residues are exposed, in contrast to the active site
triad of the β-subunit which is shielded from
solvent. This structure suggests a model in which
chorismate binding triggers a relative movement of the two domain tips of the α-subunit,
activating the β-subunit by allowing Gln to enter the active site, followed by subsequent hydrolysis. Thereby, it probably creates a channel
for passage of ammonium toward the active site
of the α-subunit.245,253.256
Trp competitively binds to a different
site than chorismate, and the competition
94
is due to conformational changes that mediate mutually exclusive binding of these
ligands. Trp blocks the arrangement that accompanies the binding of chorismate, thus
stabilizing the inactive states of both subunits.245,253,256
The Su. solfataricus AS heterotetramer
does not show cooperative binding of either
chorismate or Trp.254 This is consistent with
the lack of contact between the 2-subunits.253
ASs that display cooperative ligan-binding, such
as the ASs from Sal. typhimurium and
Ser. marcescens, have significantly different quaternary structures. Recently, the structure of an
engineered partial AS complex with Trp bound at
the regulatory site of Sal. typhimurium (1i1q.pdb),
and the structure of the AS of Se. marcescens in
the presence of its substrates (1i7q.pdb) or in the
presence of the retroinhibitor L-Trp (1i7s.pdb)
have been determined.245.256 The α-subunits of
both enzymes have a similar topology as the
α-subunit of the Su. solfataricus AS, except that
additional residues form additional β-strands and
α-helices in the Sal. typhimurium and
Se. marcescens enzymes. The quaternary structures of both the Sal. typhimurium and
Se. marcescens ASs involve extended hydrogen
bonded and hydrophobic contacts between the
catalytic subdomains of the two α-subunits, consistent with the cooperative behavior of these
enzymes. The alternative quaternary orgaizations
of the Sal. typhimurium, Se. marcescens, and Su.
solfataricus heterotetramers seem to reflect independent oligomerization events that have occurred
since the divergence of the archaebacterial and
eubacterial lineages.245,256
D. Anthranilate Synthase
Isoenzymes and Homologs
Ps. aeruginosa PAO1 contains two interchangeable AS isoenzymes. One AS, encoded
by trpE and trpG, is involved in Trp biosynthesis. The other AS, encoded by the phnA and
phnB genes, is involved in the biosynthesis of
the blue-green phenazine pigment, pyocyanin.
The LLESX10S sequence is not conserved in
PhnA, which is not retro-inhibited by Trp.257,258
Surprisingly, PhnA and PhnB are more closely
related to Es. coli TrpE and TrpG than to
Pseudomonas TrpE and TrpG, whereas
Pseudomonas TrpE and TrpG are more closely
related to Es. coli PabB and PabA (see IV.A)
than to Es. coli TrpE and TrpG.257 The phnA
gene has also been identified in Ps. aeruginosa
PNA1, where it is involved in phenazine-1carboxylate biosynthesis.259 TrpE(G) fusions,
showing low similarity to genuine ASs, have
been reported in Ps. fluorescens 2-79 (PhzE),260
Ps. aureofaciens 30-84 (PhzB),261 and in Ps.
aeruginosa PAO1 (PhzE).41,262 In strains 2–79
and 30–84, the fusion proteins were shown to
be involved in phenazine production. These
fusion proteins lack a part of the N-terminal
region, including the LLESX10S sequence, and
are probably not retro-inhibited by Trp. They
are postulated to act as ADIC synthases or to
be part of a multienzyme complex that channels anthranilate directly into the phenazine
pathway. The St. venezuelae TrpE(G) protein
displays high similarity to these hypothetical
ADIC synthases. This bears on the function of
the St. venezuelae AS, although it can complement a trpE mutant of Es. coli.205
E. Anthranilate Synthase in Plants
In plants, the Trp pathway leads to the biosynthesis of many secondary metabolites, including the auxin indole-3-acetic acid, indole
glucosinolates, anthranilate-derived alkaloids,
tryptamine derivatives, and monoterpenoid indolic
alkaloids.5,263 AS from plants was found, as in
most microbes, to consist of two dissimilar subunits.264,265 AS isoenzymes play a role in either
primary Trp biosynthesis or in secondary metabolism, and the regulation of both pathways
involves differential expression of duplicated
genes and differential retro-inhibition by Trp of
their gene products. AS genes encoding an
α-subunit and β-subunit have been cloned from
Ar. thaliana,266,267 Ruta graveolens,268 and Nicoti-
ana tabacum.269 The AS subunit α genes have
been designated ASA1/ASA2 or ASα1/ASa2. The
ASA1 or ASα1 expression is induced by wounding and/or elicitor treatment, whereas the ASA2
or ASα2 genes are expressed constitutively at low
levels.266,268
IV. ADC SYNTHASE
A. Introduction
p-Aminobenzoate (PAB) is an essential component of dihydrofolate, which in
various forms participates in, for instance,
the synthesis of purines, thymidylate,
formylmethionyl-tRNA, methionine, glycine, and panthotenate. As the folate moiety is not consumed during the reactions,
the de novo synthesis requirements for
dihydrofolate and its precursors are small.
H2-folate is synthesized from the precursors 6-hydroxymethyl-H2-pterin pyrophosphate, PAB, and Glu. As seen in Figure 6,
the conversion of chorismate to PAB requires the same regio-specific amination/
aromatization sequences as the synthesis
of anthranilate (o-aminobenzoate); however, in this case it occurs with overall
retention of position and stereochemistry. 241 Moreover, the synthesis of PAB
occurs in two steps and requires an additional enzyme.270 In a first step, chorismate
and Gln are converted to 4-amino-4deoxychorismate (ADC) and Glu. This
reaction is catalyzed by ADC synthase,
which consists of two dissimilar subunits.
pabB codes for the α-subunit with ADC
synthase activity, while pabA codes for
the β-subunit, which acts as a GATase.
PabA and PabB are highly similar to TrpG
and TrpE, respectively, suggesting they have
evolved from a common ancestor. In an
additional step, ADC is converted to PAB
and pyruvate by the pabC-encoded ADC
pyruvate-lyase. This enzyme has no counterpart in anthranilate biosynthesis.271-273
95
FIGURE 6. The formation of p-aminobenzoate (PAB) from chorismate via aminodeoxychorismate (ADC) by
the action of the three enzymes PabA, PabB, and PabC and its involvement in primary and secondary
metabolism. (This figure was adapted from Roux and Walsh.295)
B. Gene Organization and
Regulation
In Es. coli, the pabA, pabB, and pabC
genes are unlinked on the chromosome.15,16
In Ba. subtilis all the pab genes (pabB, trpG,
pabC) are present in a folate operon.171,274 In
Streptomyces lividans the pabA and pabB
genes were found to be adjacent and cotranscribed.275 In Sac. cerevisiae a bifunctional ADC synthase fusion protein is encoded by an ABZ1 or PABA(B) fusion gene,
which is located on chromosome XIV.276
The pabB gene from Es. coli codes for a
453 amino acid polypeptide with a molecular
mass of approximately 51 kDa.273 The gene
product shows 26% similarity to the Es. coli
trpE gene product. PabB is responsible for
the amination of chorismate to form the
dihydroaromatic intermediate ADC for which
it can use NH3 as amino donor.136,241,271,277
However, Es. coli pabA mutants require at
least 100 mM NH4+ for growth, compared
with only 2 mM NH4+ for a trpG mutant.278
This is consistent with the KM value of PabB
for NH3 being approximately 140 mM in the
absence of PabA, and 360 mM in the presence of PabA. This suggests that free NH3 is
not a physiological substrate for PabB.279
96
Although the N-terminal LLESX10S sequence,
involved in retro-inhibition of AS by Trp, is
conserved as LL-H/E-S-X10-D/S, no retroinhibition of ADC synthase activity by PAB
or other compounds related to folate synthesis could be demonstrated.279 The pabB gene
has also been isolated from Se. marcescens,280
Sal. typhimurium,280 and La. lactis.281
The Es. coli pabA gene encodes the 21.7kDa β-subunit of ADC synthase. The deduced amino acid sequence shows 44%
amino acid identity to AS subunit β.282 Both
GATases are highly similar but not interchangeable in Es. coli. However, in some
organisms, such as Ba. subtilis, Acinetobacter
calcoaceticus and Comamonas acidovorans,
a single gene encodes an amphibolic GATase
subunit, involved in both anthranilate and
ADC synthesis.283-287 Expression studies revealed that pabA from Es. coli is expressed
constitutively as a monocistronic transcript.
However, residual expression from a transcript including fic, the gene upstream of
pabA of which the gene product is involved
in cell division, has also been reported.288,289
Additional pabA genes have been isolated
from other enteric bacteria, such as Sal.
typhimurium, Klebsiella aerogenes, and Se.
marcescens.290
pabC codes for a 29.7-kDa polypeptide,
which shows homology to a number of Dand L-branched amino acid transaminases.291
It catalyzes the elimination of pyruvate from
ADC and the subsequent aromatization to
yield PAB.271 Although ADC pyruvate-lyase
catalyzes a reaction similar to a portion of
the reaction catalyzed by the trpE gene product, Es. coli PabC exhibits no similarity to
Es. coli TrpE. Chorismate pyruvate-lyase (see
Section VI.A) catalyzes a very similar reaction as the ADC pyruvate-lyase, except that
the substrate chorismate contains a hydroxyl
group where ADC contains an amino group.
Nevertheless, there is no significant similarity between the two enzymes. The ADC pyruvate-lyase enzyme is active as a dimer and
was shown to contain pyridoxal-5′-phosphate
(PLP) as a cofactor.272,291 Immediately downstream of pabC, a possibly co-transcribed
ORF was present, encoding a putative membrane-bound protein.291 No further work on
the expression of the gene has been reported.
Very recently, the three-dimensional structure of ADC pyruvate-lyase (1et0.pdf) from
Es. coli has been determined,292 and it was
shown that ADC pyruvate-lyase also catalyzes the transamination between D-alanine
and PLP to produce pyruvate and pyridoxamine phosphate.293 The pabC gene has also
been isolated from Ba. subtilis, where it is
part of a folate operon.171
Putative PabA, PabB, and PabC proteins
have also been identified in a large number
of organisms, based on the deduced amino
acid sequence of their genome sequences.9,10
C. Protein Interactions, Active Site
Residues, and Reaction Mechanism
The interaction between ADC synthase
subunits α and β is weak when compared
with the binding of the AS subunits. This
makes it very difficult to purify the ADC
synthase complex. By preincubation at 37°C
in the presence of 5 mM Gln, Rayl et al.278
could successfully purify the Es. coli complex, which appears to be an αβ-heterodimer.
The elution of complexed PabB and PabA
was unaffected by the presence of PabC,
suggesting that PAB synthesis does not require a higher-order structure. Despite the
weak interaction between PabB and PabA,
subunit interaction is important for both subunit activities.279 PabB has aminating activity in the absence of Gln and PabA, but the
presence of PabA stimulated the rate of conversion about fourfold. PabA is a conditional
glutaminase, only showing activity in complex with PabB, as could be demonstrated
by the use of 14C-Gln and the Gln analog
diazooxonorleucine. This indicates that the
active site of PabA is in an inactive conformation, unable to initiate nucleophilic attack
on Gln, until PabB induces a conformational
change. The addition of chorismate again
increases glutaminase activity, reflecting
another subtle conformational effect. This
indicated an ordered bi-bi mechanism for
the ADC synthase complex, in which
chorismate binds first, followed by Gln,
analogous to the mechanism determined for
AS.279,294
Mutational analysis of PabB revealed
different mutants that fell into three categories according to their properties: deficiency
of chorismate amination coupled with failure to associate with PabA, deficiency of
chorismate amination with retention of PabA
association, and competency of chorismate
amination with failure of PabA association.278
As such, the role of some residues in catalytic activity has been determined. Ser322 of
PabB is essential for ADC synthase activity.
This is consistent with a role in the nucleophilic attack at the C-2 position of chorismate,
followed by attack of exogenous ammonium
at C-4 with displacement of the nucleophile.
However, the reaction mechanism probably
operates by a significantly different mechanism when compared with AS. Sequential
97
1,3-substitution reactions have been proposed.242
The β-subunit of ADC synthase belongs,
as the β-subunit of AS, to the triad GATases,
where three conserved residues were proposed to act as a catalytic triad. However,
mutational analysis of these residues in Es.
coli (Cys79, His186, and Glu170) indicated
that only Cys79 is an essential residue for
GATase activity.295 The corresponding Cys84
of the β-subunit of the Se. marcescens AS
has also been identified as the catalytic nucleophile.249 Thus, the anticipated glutaminase mechanism of PabA, in complex with
PabB, is hydrolysis via a γ-glutamyl-Scysteinyl-enzyme intermediate.
tein showed the highest homology to the amino
acid sequence of the St. griseus PabA(B) fusion protein.133,299 The synthesis of PAB in St.
griseus was repressed by aromatic amino acids
and PAB but not by anthranilate.300 In St.
pristinaespiralis a papA gene encodes a
PabA(B) fusion and is located in a pap cluster
involved in pristinamycin biosynthesis. Disruption of papA resulted in a complete loss of
antibiotic production, but did not lead to PAB
auxotrophy.135 The ADC fusion protein from
Sac. cerevisiae, however, is not involved in
secondary metabolism as the disruption of the
encoding ABZ1 gene yielded PAB auxotrophy.276
V. ISOCHORISMATE SYNTHASE
D. Role in Secondary Metabolism
A. Introduction
Next to its importance in primary metabolism as precursor for folate, ADC is also an
intermediate in the secondary metabolic pathway for the biosynthesis of the antibiotics candicidin, chloramphenicol, and pristinamycin I
in St. griseus,296 St. venezuelae,297 and St.
pristinaespiralis,135 respectively. In these streptomycetes there is evidence for two sets of
genes directing ADC synthesis, one for primary and the other for secondary metabolism.298
The ADC synthase, involved in secondary
metabolism, appears to be a fusion protein.
This fused ADC synthase of St. venezuelae is
encoded by a pabA(B) fusion gene, which codes
for a 670 amino acid polypeptide, of which the
N- and C-terminal regions resemble PabA and
PabB, respectively, from other microorganisms.134 The disruption of the chromosomal
copy of the cloned pabA(B) of St. venezuelae
did not cause auxotrophy, but virtually eliminated synthesis of antibiotic. This confirms the
presence of a second ADC synthase isoenzyme involved in folate biosynthesis.134 Indeed, a second set of pabA and pabB genes has
already been identified in St. venezuelae
(AAF01062 and AAF01063). The fusion pro-
98
Isochorismate synthase (ICS or isochorismate
hydroxymutase; EC 5.4.99.6) catalyzes the conversion of chorismate to isochorismate.
Isochorismate forms a branch point substrate as
the precursor for both menaquinone and some
siderophores. Isochorismate is highly unstable
and under normal growth conditions the intracellular concentration of chorismate prevails significantly over isochorismate.301,302 Isochorismate is
required under very different environmental conditions: siderophores are produced mainly under
aerobic conditions of iron deprivation, while
menaquinone serves in Es. coli as a component of
the anaerobic electron transport chain.
1. Siderophores
Iron is prevalent in the environment as a
component of highly insoluble ferric hydroxides and is in biological systems chelated by
high-affinity iron binding proteins.303 Most
microorganisms have developed highly specific iron assimilation pathways based on
the secretion of low-molecular-weight high-
affinity chelators, termed siderophores, and
the subsequent transport of the ferric complex of these molecules.304 For example, Es.
coli and other enteric bacteria produce in
conditions of iron deprivation a very strong
chelator, enterobactin.301,305 Enterobactin is
a cyclic triester of dihydroxybenzoylserine
and its biosynthesis is conveniently divided
into two parts: (1) the conversion of
chorismate to the specific precursor
2,3-dihydroxybenzoate (DHB), and (2) the
assembly of enterobactin by a nonribosomal
peptide synthase in which three molecules
of DHB are condensed with three molecules
of serine (Figure 7).11,255,306,307 The synthesis
of DHB also involves the action of an
isochorismate hydroxymutase, encoded by
entB, which converts isochorismate to
2,3-dihydro-2,3-dihydroxybenzoate. However, this protein can also use chorismate
as a substrate to form 3,4-dihydro-3,4dihydroxybenzoate, which is presumably an
adventitious reaction.302,308
2. Quinones
Quinones are lipophilic, non-protein
components of the membrane-bound electron-transfer chain in both prokaryotes and
eukaryotes. They can be divided into two
major structural groups: benzoquinones and
naphthoquinones. The benzoquinones are
termed ubiquinones (coenzyme Q; UQn), and
the naphthoquinones are termed either
menaquinones (Vitamin K 2 ; MQ n ) or
demethylmenaquinones (DMQn). The n refers to the length of the isoprenoid side chain.
Most Gram-positive bacteria and anaerobic
Gram-negative bacteria possess only
menaquinones, whereas the majority of
strictly aerobic Gram-negative bacteria contain ubiquinone exclusively. Both types of
isoprenoid quinones are only found in facultative anaerobic Gram-negative bacteria.309
The major quinones in Es. coli are UQ8,
MQ8, and DMQ8. The ratio of the quinones
is variable and depends on the growth conditions. Aerobically grown Es. coli cells contain more UQ8 than MQ8 and DMQ8, whereas
in anaerobic cells this profile is reversed.310
MQ7 is the predominant isoprenoid homolog
found in Bacillus spp.311,312 The biosynthesis
of menaquinone requires the formation of a
naphtoquinone ring via a series of reactions
branching from the shikimate pathway.
Menaquinone-specific reactions catalyze the
formation of o-succinylbenzoate from
chorismate, which is converted to 1,4dihydroxy-2-naphtoate, by utilizing an
o-succinylbenzoate-coenzyme A intermediate (Figure 7).313,314
B. Organization and Regulation
Both model organisms Es. coli and Ba.
subtilis contain two ICS isoenzymes. In Es.
coli two differentially expressed genes, entC
and menF, code for ICSs involved in DHB
and menaquinone biosynthesis, respectively.
The Ba. subtilis homologs are called dhbC
and menF, respectively.
1. Escherichia coli
The enterobactin system in Es. coli consists of four polycistronic transcripts expressed from two bidirectional iron-repressive control regions.15,315,316 One of these
transcriptional linkages is the entCEBA cluster.308 The entC gene codes for a 391 amino
acid polypeptide with a calculated molecular mass of 42.9 kDa.255 The deduced amino
acid sequence shows in the C-terminal part
similarity to other chorismate-utilizing enzymes, such as AS and ADC synthase.255
The promoter of the entCEBA operon overlaps with an operator containing two Furrepressor binding sites. The fur gene product
acts as a dimer with ferrous iron as co-re-
99
100
FIGURE 7. Branched pathway in Es. coli leading to menaquinone and enterobactin. The menaquinone pathway is encoded by the men genes
and routes via o-succinylbenzoate (OSB) and 1,4-dihydroxy-2-naphtoate (DHN). The enterobactin pathway is encoded by the ent genes and
routes via 2,3-dihydro-2,3-dihydroxybenzoate (DHDHB) and 2,3-dihydroxybenzoate (DHB).
pressor via sequence-specific protein-DNA
interactions at the promoter regions (ironbox) of Fur-controlled genes.255,301,315,317 Using an entC-lacZ fusion, it was established
that entC is expressed and regulated by iron
both aerobically and anaerobically.305 The
possible role of anaerobic enterobactin production remains to be determined. An entC
mutant still produces menaquinone under
anaerobic conditions in iron-sufficient media but no enterobactin.305,317 The EntC protein functions as a monomer and catalyzes
the Mg 2+-dependent interconversion of
chorismate and isochorismate in both directions with a KM value for chorismate of
14 µM and for isochorismate of 5 µM.301,318
A dual role for EntC in both enterobactin
and menaquinone biosynthesis was postulated,319 until on the basis of regulatory studies evidence against the involvement of the
entC gene in menaquinone synthesis was
obtained.305 This led to the isolation of a new
ICS of Es. coli, encoded by menF, which is
specifically involved in menaquinone biosynthesis.320,321 Seven men genes involved in
the synthesis of menaquinone have been identified.314 The menF gene is clustered with
other menaquinone genes and overlaps 114
bp with the downstream menD gene. The
menF gene codes for a 430 amino acid protein with a calculated molecular mass of
48.7 kDa and exhibits a 24% overall identity
with EntC. Menaquinone biosynthesis appears to be regulated by the availability of
oxygen.320 However, the UQ/MQ ratio seems
to be posttranslationally regulated.322 In the
promoter region of menF a putative cAMP
receptor protein (CRP) binding site is present,
indicating that menF is regulated differently
from entC.317 cAMP bound to the dimeric
CRP interacts with specific sequences in
cAMP-CRP-responsive promoters and may
function as a positive or negative effector.
Under anaerobic conditions and in the presence of nonfermentable carbon sources with
fumarate as terminal electron acceptor,
menaquinone is required as an essential electron transport compound. Under these conditions cAMP levels are high, which in turn
may stimulate transcription of menF.317 An
Es. coli mutant with a disrupted menF produced enterobactin and only a trace of
menaquinone. Neither enterobactin nor
menaquinone is detectable in an entC-menF
double mutant.317
MenF was purified to homogeneity and
was found to have a molecular mass of 98
kDa. Thus, the enzyme functions as a
homodimer. It carries out the conversion of
chorismate to isochorismate in the presence
of Mg2+. The enzyme was found to have a
KM value for chorismate of 195 µM and
showed a pH optimum of 7.5 to 8.0.318 Using
purified 6× His-tagged MenF, the reverse
reaction could be observed and a KM value
for isochorismate of 119 µM was determined.317
2. Bacillus subtilis
Ba. subtilis produces the siderophore
bacillibactin, a DHB-glycine-threonine trimeric ester.323 The genes encoding the various steps for bacillibactin biosynthesis are
organized in an operon, dhbACEBF.41,324
dhbC encodes a protein of 396 amino acids
with a predicted mass of 43.8 kDa and shows
35% identity at the amino acid level with
EntC of Es. coli. DhbC can compensate for
the lack of MenF in a menF mutant. However, a dhbC mutant produced wild-type levels of menaquinone but was DHB deficient.
Transcriptional analysis showed that the
expression of dhbC is iron regulated. Mutations in the iron-box sequence within the
dhb promoter region abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in
Ba. subtilis.311
The Ba. subtilis menF gene forms an
operon with menD.325 menF codes for a 471
101
amino acid protein with a predicted mass of
51.8 kDa and shows 35% amino acid identity with the Ba. subtilis DhbC. The menp1
promoter is the primary cis-element for
menFD gene expression and is responsive to
carbon source and growth phase. Supplementation with nonfermentable carbon
sources or reutilization of glycolytic end
products increases menp1 activity in the late
exponential growth phase.311,326 Ba. subtilis
will utilize fermentable carbon sources for
complete glycolysis to glycolytic end products. Only when the fermentable carbon
sources are exhausted, does it enter an oxidative metabolic stage, which requires a coordination of the TCA cycle activity with the
formation of respiratory chain components,
especially menaquinone.326 Excretion of glycolytic end products increases the external
pH, and expression of both the menFD and
the menBE operons occurs only in acidic
conditions (pH 5.5).326,327 In the –35/–10 region of the menp1 promoter a TGAAA motif, which is present in promoter regions of
many genes encoding oxidative functions,
was found. Mutations in this TGAAA motif
resulted in unregulated menp1 activity.326,328
Regulation of the menFD operon occurs
possibly via an internal inducer molecule,
for which NADH is a good candidate, as its
cellular concentration can serve as a measure of carbon availability.326
3. Other Microorganisms
Most mesophylic Aeromonas spp. produce either enterobactin or amonabactin.
Amonabactin is synthesized in two biologically active forms, each composed of DHB
conjugated to lysine, glycine, and either Trp
(amonabactin-T) or Phe (amonabactinP).329,330 By complementation of an Es. coli
mutant, which requires DHB to support
enterobactin biosynthesis, the amoA gene of
Ae. hydrophila was isolated. An 1188-bp
102
ORF, encoding 396 amino acids, shows at
the C-terminal portion of the deduced amino
acid sequence 58% identity with the Es. coli
EntC protein. The putative –35/–10 promoter
overlaps with a putative iron-regulating sequence resembling a Fur-repressor binding
site. The amoA gene is presumably also organized in an Es. coli entCEBA-resembling
operon and an amoA Tn5 mutant excreted
neither DHB nor amonabactin.330
Vibrio cholerae secretes a catechol
siderophore vibriobactin in response to iron
limitation. Vibriobactin contains three DHB
residues and two molecules of L-threonine,
which are all linked to a backbone of
norspermidine.331,332 Vi. cholerae vib genes,
homologous to the Es. coli entA, entB, entC,
entD, and entE genes, were identified. The
vib genes are adjacent, but are organized
into a least three transcriptional units. vibC
codes for a 395 amino acid protein of 43.6
kDa. It shows identity of about 40% to the
Es. coli EntC protein and to EntC homologs
from other organisms.316 A second ICS-encoding gene has been identified in the genome sequence of Vi. cholerae.38
Azotobacter vinelandii forms different
catecholate siderophores during iron-limited
growth. The first gene, csbC, in the
catecholate siderophore biosynthesis operon
encodes an ICS. The region upstream of csbC
contains a typical –35/–10 promoter that
overlaps a Fur-box and a Sox-box. Expression analysis of csbC revealed dual regulation by iron and oxidative stress.333
Salicylate (o-hydroxybenzoate) is a highaffinity siderophore and is the precursor of another siderophore pyochelin in Ps. aeruginosa.
The pchDCBA operon of Ps. aeruginosa is required for the synthesis of salicylate from
chorismate and the activation of salicylate for
further synthesis of either pyochelin or the antifungal antibiotic dihydroaeruginoate.334 The
PchA protein is predicted to consist of a 476
amino acid polypeptide with a calculated molecular mass of 52 kDa.335 The deduced amino
acid sequence shows extensive similarity to the
known ICSs. PchB is a 102 amino acid protein
that shows marginal homology to chorismate
mutases. In the production of salicylate, pyruvate is formed in equimolar amounts. This leads
to the following proposed pathway for salicylate biosynthesis in Ps. aeruginosa: chorismate
is converted to isochorismate by PchA, and in a
second step salicylate and pyruvate are formed
by isochorismate pyruvate-lyase activity of PchB.
Salicylate synthesis in Ps. aeruginosa is repressed by iron as the transcription of the
pchDCBA operon starts at promoters that overlap with two Fur boxes and the promoter of the
divergently transcribed pchR gene, encoding
the transcriptional activator of pyochelin and
ferri-pyochelin receptor synthesis.334 The ironregulated pmsCEAB gene cluster of the
biocontrol strain Ps. fluorescens WCS374, of
which the pmsC and pmsB genes are homolgous
to the Ps. aeruginosa pchA and pchB genes,
respectively, is involved in the biosynthesis
of salicylate and the siderophore pseudomonine.336 A pchB homologous gene was also
identified in Vi. vulnificus.337
The genes involved in the formation of
the salicylate moiety in mycobactin
siderophores of Mycobacterium tuberculosis are not clearly defined.338 It was suggested that a distinct operon with two
genes, entC and entD, homologous to those
implicated in salicylate formation in
Ps. aeruginosa, is responsible for its formation.339 Quadri et al.340 concluded that
the product of another gene, mbtI, which
is homologous to AS, might be necessary
for converting chorismate directly to
salicylate. The ybtS- and irp9-encoded gene
products of Ye. pestis and Ye. enterocolitica, respectively, show high similarity to MbtI of My. tuberculosis and are
postulated to be involved in the formation
of salicylate for the biogenesis of the
siderophore yersiniabactin.306,341
ICS-encoding genes have also been identified in the complete genome sequence of
Ha. influenzae Rd,32 Halobacterium sp.
NRC-1,69 and Synechocystis sp. PCC6803.73
C. Reaction Mechanism
ICS catalyzes a stereo- and regiospecific
1,5 double-SN2′ displacement of the 4-hydroxyl
group in chorismate with a hydroxyl group at
the C6 position.11 The reaction has an absolute
requirement for Mg2+ and is reversible, favoring chorismate. Labeling experiments with
H218O in both Es. coli and Kl. pneumoniae
established that the incoming hydroxyl group
during the reaction comes from water rather
than from O2 or via intramolecular hydroxyl
transfer.301,342 ICS displays sequence homology with AS and ADC synthase.255 Because
the reaction does not require an amide transfer,
there is no need for a function analogous to the
GATase component of AS and ADC synthase.304 ICS catalyzes a very similar reaction
as AS, except that the incoming nucleophile is
water instead of ammonium and that it does
not catalyze the further aromatization of the
resultant isochorismate.11 The similarity between ICS and AS was extended by the finding
that ICS, like AS, catalyzes the formation of
ADIC in the presence of ammonium, confirming that they are functionally related.242 By
using specifically designed inhibitors, it was
shown that ICS, as AS, catalyzes a concerted
syn-1,5 displacement via a Mg2+-bound transition state (Figure 8).242 All the above observations were done with the Es. coli EntC protein.
However, the Es. coli MenF protein probably
catalyzes the same 1,5 double-SN2′ displacement with water, as it also requires Mg2+, and
menaquinone is synthesized in mainly anaerobic conditions.318
D. Isochorismate Synthase in Plants
In plants, chorismate is partitioned over
various primary (menaquinones and phyllo-
103
FIGURE 8. Proposed concerted 1,5 double-SN2’ displacement mechanism for the ICS reaction. It involves a
Mg2+-bound chair-like intermediate. (This figure was adapted from Walsh et al.11)
quinones) and secondary (anthraquinones,
naphtoquinones, and certain alkaloids) pathways.343 In higher plants little work has been
done on ICS. The enzyme has been purified
partially from Gallium mollugo, while in
Catharanthus roseus the enzyme was purified to homogeneity and the corresponding
cDNA was cloned.344,345 In Cat. roseus two
ICS isoforms were found after elicitation
with fungi. However, Southern analysis indicated the existence of only one ICS gene,
suggesting posttranscriptional modification. The deduced amino acid sequence of
the C-terminal region shows 30% identity
with bacterial ICSs and 30% similarity with
component I of AS in plants. The protein
has an N-terminal chloroplast-targeting
signal. Both isoforms do not differ much
in their biochemical properties and both
show a low affinity for chorismate when
compared with that of AS.264,345 In Ar.
thaliana, two putative ICS genes have been
sequenced.346,347
104
In plants salicylate plays a central role in
defense against pathogen attack. The overproduction of salicylate in Ar. thaliana, by
the expression of an engineered Ps.
aeruginosa salicylate synthase pchB(A) fusion, and in tobacco, by transgenic expression of the Es. coli entC and the
Ps. fluorescens pmsB genes, enhances pathogen resistance.348-350
VI. CHORISMATE PYRUVATELYASE
A. Introduction
Chorismate pyruvate-lyase catalyzes the
conversion of chorismate to p-hydroxybenzoate (PHB). PHB is a key intermediate in the biosynthesis of ubiquinones. In the
biosynthesis pathways of ubiquinone, the
core is derived from chorismate, whereas
the prenyl side chain is derived from prenyl
diphosphate and the methyl groups are derived from S-adenosylmethionine. The many
steps involved in ubiquinone biosynthesis
are encoded by the ubi genes in Es. coli and
COQ genes in Sac. cerevisiae.309,314 Not much
work on this chorismate-aromatizing enzyme
has been reported.
indicate that the ubiCA operon is negatively
regulated by the transcriptional regulators
Fnr and IHF in function of the oxygen availability.357 An Es. coli ubiCA mutant produces no ubiquinone and showed a severely
diminished growth rate under aerobic conditions.358
B. Gene Organization and
Regulation
C. Protein Sequence and
Characteristics
A chemically induced Es. coli AN244
mutant deficient in PHB synthesis was already isolated in 1974. 351 However,
chorismate pyruvate-lyase was the last
chorismate-utilizing enzyme to be characterized in Es. coli. The ubiC gene, coding
for chorismate pyruvate-lyase of Es. coli,
was cloned and sequenced independently
by two groups. Siebert et al.352 isolated the
gene by genetic complementation of the
Es. coli ubiC mutant, AN244. Nichols and
Green353 based their isolation on the position of the ubiA gene on the Es. coli map.354
ubiA codes for the membrane-bound PHBoctaprenyl transferase, which catalyzes the
subsequent step in ubiquinone biosynthesis.355 The ubiC and ubiA genes are organized in an ubiCA operon followed by a
putative Rho-independent terminator.352 The
promoter of the ubiCA operon has been
localized, but no sequence element with
high homology to the Es. coli consensus
promoter could be detected, indicating a
moderate strength promoter.355,356 Using an
ubiC-lacZ fusion it was shown that the expression of the ubiCA operon was higher
aerobically than anaerobically and increased
with the rate of oxygen supply. Mutations
in fnr (fumarate nitrate regulation) and himA,
encoding the integrating host factor (IHF),
enhanced the anaerobic expression of
ubiCA. Analysis of the promoter region of
the ubiCA revealed putative, overlapping
binding sites for Fnr and IHF. These results
ubiC from Es. coli codes for a 165 amino
acid polypeptide, of which the N-terminal
formyl-methionine is removed. The amino
acid sequence shows no significant similarities to other proteins, including the other
chorismate-utilizing enzymes, and ADC
pyruvate-lyase (PabC).352 However, overproduction of UbiC in Es. coli can complement a pabC mutation, indicating functional
similarity.353 UbiC has a calculated molecular mass of 18.7 kDa and the native enzyme
is active as a monomer.353 It is the only
soluble enzyme of the ubiquinone biosynthetic pathway.309,310 The pH optimum of
purified chorismate pyruvate-lyase is 7.5,
and it has an apparent K M value for
chorismate of 6.1 µM. The enzyme activity
does not require metal cofactors and no
reverse reaction could be observed.353,356
Although chorismate pyruvate-lyase activity does not appear to require added ATP in
vitro, the presence of ATP-binding ‘motif
A’ was reported.355 However, this sequence
is not conserved among the putative
chorismate pyruvate-lyases (see below).
Chorismate pyruvate-lyase activity is probably posttranslationally regulated as the
enzyme is subject to competitive retro-inhibition by PHB.356
Similar protein sequences can be found
in the translated genome sequences of
Es. coli O157:H7,16 Vi. cholerae, 38 and
Ps. aeruginosa41 and has been isolated from
Ps. putida.359
105
D. Crystal Structure and Reaction
Mechanism
A fully active double mutant in which
the two Cys residues were replaced by two
Ser residues of chorismate pyruvate-lyase
from Es. coli was crystallized. It diffracts to
1.1 Å resolution and awaits further characterization.360
It has been proposed that the aromatization step occurs via a 1,2-elimination, starting with an initial abstraction of the C4 hydrogen of chorismate followed by the loss of
the C5-enolpyruvyl group (Figure 9).11 Sequencing of the ubiC gene of the Es. coli
AN244 ubiC mutant revealed a G to A transition resulting in a change from Glu156 to
Lys.356 The negative charge of Glu156, which
is completely conserved among the putative
chorismate pyruvate-lyases, may be essential for the enzymatic mechanism as Glu can
serve in the first step as a nucleophilic group
for acceptance of the proton. The proton
needed for the pyruvate abstraction in the
second step could be supplied by a basic
residue, for example, Lys or Arg. The puri-
fied chorismate pyruvate-lyase enzyme did
not accept isochorismate (C6-OH) as substrate, but, on the other hand, could convert
ADC to PAB. Therefore, a hydroxy or amino
function in the 4-position appears to be essential for the reaction.353,356
E. Alternative p-Hydroxybenzoate
Biosynthesis Pathways
Corynebacterium cyclohexanicum generates
PHB from p-oxocyclohexane carboxylate.361
Feeding experiments with labeled shikimate in
Es. coli showed, however, that chorismate pyruvate-lyase is the sole enzymatic source of PHB
in vivo in Es. coli.356 Higher plants, in contrast,
produce PHB via the phenylpropanoid pathway, and the conversion of chorismate to PHB
in plants involves up to 10 successive enzymatic
reaction steps. It was shown that in Lithospermum
erythrorhizon cell cultures p-coumarate is a precursor of PHB.362
The ubiC gene of Es. coli was used for
genetic engineering of plant secondary metabolism. The introduction of the ubiC gene
FIGURE 9. Hypothetical 1,2-elimination reaction mechanism of the aromatization of chorismate to PHB by
chorismate pyruvate-lyase involving a Glu residue and a basic residue. (This figure was adapted from Siebert
et al.356)
106
in tobacco led to an accumulation of PHBglucosides,363,364 and ubiC transformed Li.
erythrorhizon showed chorismate pyruvatelyase activity.365
VII. EVOLUTIONARY ASPECTS
Comparison of the amino acid sequences
of the chorismate-utilizing enzymes indicates
that AS, ICS, and ADC synthase share significant sequence similarity, in contrast to
CM and chorismate pyruvate-lyase.
CM comprises a set of CM isoenzymes,
which, intriguingly, are very divergent at
their amino acid level. However, the amino
acids that contribute to their active site are
conserved. The AroQ class proteins, from
both prokaryotes and eukaryotes, display
tertiary structures that are clearly related. In
particular, the helix-bundle topology and
crucial catalytic residues are conserved. This
indicates that the AroQ CMs evolved from a
common ancestor. The low conservation of
this ancestry on the amino acid level may be
due to the lack of functional pressure as the
reaction mechanism in mechanistic terms is
presumably very simple. Evolutionary drift
of some members of the AroQ family may
have been influenced by the nature of its
fusion to attached functional domains or by
the location of the protein in the cell. Moreover, the acquisition of allosteric regulation
in the case of yeast CM required significant
divergence of the primary structure.13 It is
also conceivable that some of the AroQ proteins have diverged in function during evolution. For example, the ADC mutases from
Streptomyces spp. catalyze a similar reaction as CM, except that their substrates differ in the para-position. The AroH CM from
Ba. subtilis has a completely different threedimensional structure. Interestingly, the active sites of both enzyme classes are similarly functionalized, suggesting that they
arose by a process of convergent evolution.
These findings reinforce the notion that a
function can be evolutionary conserved via a
common mechanism, rather than via sequential or structural homology.109
The C-terminal 250 residues of the AS
α-subunit show similarity to both ADC synthase and ICS. The three enzymes catalyze
the conversion of chorismate to ADIC, ADC,
and isochorismate, respectively. ADIC is an
isomer of ADC and is the amino analog of
isochorismate. Only AS is a bifunctional
enzyme that catalyzes the subsequent lyase
reaction of ADIC to anthranilate. Structural
similarity suggests common ancestry and/or
related functionality. Structural and functional relationship of AS and ADC synthase
is further testified by the fact that in some
organisms both proteins use the same
GATase subunit.3 In addition, antibodies
raised against the AS α-subunit cross-react
with the ADC synthase α-subunit.366 It is
striking that conservation of amino acids
occurs mainly in the C-terminal portion of
the AS, ICS, and ADC synthase proteins.
Thus, it was proposed to have C-terminal
conservation for the reaction and N-terminal
divergence for regulation.255 However, the
recent elucidation of the crystal structure of
the Su. sulfataricus AS rules out the distinction between an N-terminal regulatory and a
C-terminal catalytic domain.253 This could
indicate that especially structural and functional features related to substrate recognition and binding may be conserved, and that
their enzymatic reaction requirement may
be quite distinct. However, CM and
chorismate pyruvate-lyase also utilize
chorismate as a substrate, but they are not
similar in sequence. So, it is more likely that
the chorismate-binding subunits of AS, ADC
synthase, and ICS are evolutionarily related.
Kozlowski et al.242 investigated the reaction
mechanism of the three enzymes that all
require Mg2+ as a co-factor. They confirmed
a number of similarities and differences between the three related enzymes. ICS and
107
ADIC synthase are mechanistically very
similar, both working via a 1,5-substitution
mechanism, the incoming nucleophile being
either a hydroxyl group or ammonium, respectively. This was confirmed by the finding that ICS, like AS, catalyzes the formation of ADIC in the presence of ammonium.
ADC synthase catalyzes the substitution of
the 4-hydroxyl group of chorismate with
ammonium but with retention of position
and stereochemistry, presumably by a significantly different mechanism. Gene duplication is a major mechanism for the increase
of genetic potential and metabolic diversity.
In the case of ADIC synthase, ICS, and ADC
synthase, the duplication of an ancestral gene
and the divergence of residues involved in
catalytic specificity, probably led to the derivation of enzymes with similar catalytic capabilities. As a consequence, these enzymes
should be denoted as paralogs.367
Such a duplication-divergence model
would be expected to account for the relationship between ADC pyruvatelyase, chorismate pyruvate-lyase, and
isochorismate pyruvate-lyase. These enzymes catalyze the elimination of an
enolpyruvyl moiety concomitantly with aromatization of the ring structure. Their substrates differ only in the presence of a
4-amino and 4-hydroxy vs. a 2-hydroxy
group, respectively. Moreover, by complementation it was shown that chorismate
pyruvate-lyase can use ADC as a substrate.353 However, the proteins show no
significant sequence homology. ADC pyruvate-lyase even appears to be unique among
the enzymes that utilize chorismate or one
of its derivatives as a substrate, as it requires PLP as a cofactor.291 The reaction
mechanism of ADC pyruvate-lyase via a
Shiff base, consequently, differs strongly
from the other lyase mechanisms. The enzymes appear to represent an evolutionary
convergence toward similar reaction capabilities from different ancestral progenitors.
108
VIII. REGULATION OF THE
CHORISMATE POOL
The regulation of a biosynthetic pathway affects in most cases the first step of the
pathway by modulating gene expression and/
or enzyme activity. The enzymes that initiate the various metabolic pathways from
the branch point chorismate therefore may
play a key role in cellular aromatic metabolism and in the regulation of the distribution
of this precursor over the various pathways.
It is also of key importance for a cell to
synthesize chorismate in adequate concentrations under various environmental conditions. For the correct function of a metabolic
node, the control of transcription, translation, and enzyme activity are essential interwoven regulatory systems.368 Moreover, fine
regulation of the shikimate pathway is essential as the pathway is energy consuming.
For example, the aromatic amino acids are
energetically the most costly amino acids for
the living cell. Because microorganisms use
more than 90% of their metabolic energy for
protein biosynthesis, for most prokaryotes,
the three aromatic amino acids represent
nearly the entire output of aromatic biosynthesis, and regulatory mechanisms for
shikimate pathway activity are triggered by
the intracellular concentrations of Phe, Tyr,
and Trp.6
A. Escherichia coli
In Es. coli, the synthesis of chorismate is
regulated in function of the concentration of
the aromatic amino acids. Of all the genes of
the prechorismate shikimate pathway, only
the three genes specifying the three DAHP
synthases and the gene for shikimate kinase
II are subject to control. The first step of the
shikimate pathway is the conversion of erythrose 4-phosphate and phosphoenolpyruvate
to DAHP by three DAHP synthase isoen-
zymes. DAHP synthaseTyr is subject to retroinhibition by Tyr and the encoding aroF
gene is part of the TyrR regulon. DAHP
synthaseTrp is subject to retro-inhibition by
Trp and the corresponding aroH gene is part
of the TrpR regulon. DAHP synthasePhe is
retro-inhibited by Phe, and the encoding aroG
gene is probably repressed by Phe and Trp
via TyrR. Thus, each amino acid influences
the flow of carbon into the pathway. Also,
two shikimate kinase enzymes are present.
The aroL gene, encoding shikimate kinase
II, is highly regulated as it is part of both the
TyrR and TrpR regulons. It has been shown
that in the presence of all three aromatic
amino acids, the cell retains enough residual
activity of either DAHP synthasePhe or DAHP
synthaseTrp to allow continued synthesis of
chorismate, sufficient for the synthesis of
the aromatic vitamins.369,370
The activity of the five chorismate-utilizing enzymes in Es. coli is highly regulated. The biosynthesis of the aromatic amino
acids can be regulated at the transcriptional
level, but also by retro-inhibition. This enables the cell to modulate aromatic amino
acid biosynthesis without de novo protein
synthesis, for which the amino acids are required. The AS genes are regulated by Trp
via attenuation and repression, while the AS
enzyme is subject to retro-inhibition by Trp.
The two CMs, P- and T-proteins, are retroinhibited by Phe and Tyr, respectively. The
encoding genes, aroQp.pheA and aroQt.tyrA,
are regulated by Phe via attenuation and by
Tyr via the TyrR regulator. The two ICS
encoding genes, entC and menF, are regulated in function of the iron concentration
and the oxygen status of the cell, respectively. The ubiC gene encoding chorismate
pyruvate-lyase is expressed aerobically. No
regulation of the ADC synthase activity has
been established yet. The absence of regulation of the ADC synthase activity can be
explained by the fact that tetrahydrofolate is
required as a donor of one-carbon units in a
variety of biosynthetic processes and under
all environmental conditions.
Because these enzymes compete with
each other for the same substrate, it is of
interest to compare the affinities of each for
chorismate. The reported KM values for
chorismate are 1.2 µM (TrpE);371 45 µM
(AroQp.pheA);372 92 µM (TyrA);45 14 µM
(EntC);301 195 µM (aroQt.MenF);318 4.2 µM
(PabB),279 and 6.1 µM (UbiC).356 This very
marked difference in affinities implies
that under conditions of chorismate limitation, chorismate would be preferentially directed down the Trp pathway, rather than
toward Phe or Tyr. Accordingly, the AS
enzyme lacks Trp residues, while the other
chorismate-utilizing enzymes contain this
amino acid. Hence, the aromatic metabolism
is diverted to Trp production in Trp starvation conditions.
B. Bacillus subtilis
Ba. subtilis uses a different strategy to
regulate the flow of metabolites into the
common aromatic amino acid pathway. Here,
prephenate plays a central role in the regulation. In Ba. subtilis there exist two different
DAHP synthases. One monofunctional
DAHP synthase is inhibited by prephenate.
The other DAHP synthase is fused to CM
and is sequentially retro-inhibited by
chorismate and prephenate. Moreover, the
encoding gene is moderately repressed by
Tyr. Shikimate kinase is active in a complex
with DAHP synthase/CM and is also inhibited by prephenate. As the prephenate
dehydratases and the prephenate dehydrogenases are retro-inhibited by Phe and Tyr,
respectively, excess of Phe and Tyr will accumulate prephenate, which in turn reduces
the flow of precursors into the common
shikimate pathway. Also, CM is product
inhibited by prephenate. It has been proposed that as a consequence, chorismate is
109
sufficiently shuttled by AS into the Trp pathway. AS is retro-inhibited by Trp and its
expression is regulated by Trp via TRAP.
The high KM value of CM for chorismate
(100 µM) probably also directs chorismate
to AS in chorismate-limited conditions.61,64
C. Saccharomyces cerevisiae
In yeast, two differently regulated DAHP
synthases are encoded by the genes ARO3 and
ARO4 and are inhibited by Phe (Ki 75 µM) and
Tyr (Ki 0.9 µM), respectively. The Ki values of
the two isoenzymes indicate a major flux toward Phe in the aromatic amino acid
biosythesis. Both genes are regulated by the
transcriptional activator GCN4.373-376 Basal
level expression of ARO3 is additionally
upregulated by the global factor ABF1 and
repressed through an URS1 element.377 No
Trp-regulated DAHP synthase is present in
yeast. The pool of chorismate is regulated
through CM and AS mainly by the regulation
of the enzymatic activities.108 AS is retro-inhibited by Trp, and its encoding genes are
regulated by the general amino acid control
system. The expression of CM is not regulated; however, this requires a sophisticated
allosteric regulation of its CM activity through
activation by Trp and inhibition by Tyr.368
ACKNOWLEDGMENTS
F.D. was a recepient of a grant from the
Vlaams Instituut ter Bevordering van het
Wetenschappelijk-Technologisch Onderzoek
in de Industrie (IWT) and is now a recipient
of a postdoctoral fellowship from the research council (K.U. Leuven, Belgium).
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