* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download A Metabolic Node in Action: Chorismate
Ridge (biology) wikipedia , lookup
Catalytic triad wikipedia , lookup
Magnesium transporter wikipedia , lookup
Proteolysis wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Genetic code wikipedia , lookup
Gene desert wikipedia , lookup
Community fingerprinting wikipedia , lookup
Gene nomenclature wikipedia , lookup
Metalloprotein wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Gene expression wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Point mutation wikipedia , lookup
Biochemistry wikipedia , lookup
Gene expression profiling wikipedia , lookup
Expression vector wikipedia , lookup
Gene regulatory network wikipedia , lookup
Biosynthesis of doxorubicin wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Biosynthesis wikipedia , lookup
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 75 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 79 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). REFERENCES 1. Schmid, J. and Amrhein, N., Molecular organization of the shikimate pathway in higher plants, Phytochemistry, 39, 737, 1995. 110 2. Bentley, R., The shikimate pathway: a metabolic tree with many branches, Crit. Rev. Biochem. Mol. Biol., 25, 307, 1990. 3. Romero, R. M., Roberts, M. F., and Phillipson, J. D., Anthranilate synthase in microorganisms and plants, Phytochemistry, 39, 263, 1995. 4. Romero, R. M., Roberts, M. F., and Phillipson, J. D., Chorismate mutase in microorganisms and plants, Phytochemistry, 40, 1015, 1995. 5. Radwanski, E. R. and Last, R. L., Tryptophan biosynthesis and metabolism: biochemical and molecular genetics, Plant Cell, 7, 921, 1995. 6. Herrmann, K. M., The shikimate pathway as an entry to aromatic secondary metabolism, Plant Physiol., 107, 7, 1995. 7. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J. H., Zhang, Z., Miller, W., and Lipman, D. J., Gapped BLAST and PSIBLAST: a new generation of protein database search programs, Nucleic Acids Res., 25, 3389, 1997. 8. Thompson, J. D., Higgins, W., and Gibson, T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice, Nucleic Acids Res., 22, 4673, 1994. 9. Tatusov, R. L., Koonin, E. V., and Lipman, D. J., A genomic perspective on protein families, Science, 278, 631, 1997. 10. Overbeek, R., Larsen, N., Pusch, G. D., D’Souza, M., Selkov, E. J., Kyrpides, N., Fonstein, M., Maltsev, N., and Selkov, E. , WIT: integrated system for high-throughput genome sequence analysis and metabolic reconstruction, Nucleic Acids Res., 28, 123, 2000. 11. Walsh, C. T., Liu, J., Rusnak, F., and Sakaitani, M., Molecular studies om enzymes in chorismate metabolism and the enterobactin biosynthetic pathway, Chem.Rev., 90, 1105, 1990. 12. Gu, W., Williams, D. S., Aldrich, H. C., Xie, G., Gabriel, D. W., and Jensen, R. A., The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison, Microb.Comp.Genomics, 2, 141, 1997. 13. MacBeath, G., Kast, P., and Hilvert, D., A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii, Biochemistry, 37, 10062, 1998. 14. Davidson, B. E., Chorismate mutaseprephenate dehydratase from Escherichia coli, Methods Enzymol., 142, 432, 1987. 15. Blattner, F. R., Plunkett, G., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y., The complete genome sequence of Escherichia coli K-12, Science, 277, 1453, 1997. 16. Perna, N. T., Plunkett, G., Burland, V., Mau, B., Glasner, J. D., Rose, D. J., Mayhew, G. F., Evans, P. S., Gregor, J., Kirkpatrick, H. A., Posfai, G., Hackett, J., Klink, S., Boutin, A., Shao, Y., Miller, L., Grotbeck, E. J., Davis, N. W., Lim, A., Dimalanta, E. T., Potamousis, K. D., Apodaca, J., Anantharaman, T. S., Lin, J., Yen, G., Schwartz, D. C., Welch, R. A., and Blattner, F. R., Genome sequence of enterohaemorrhagic Escherichia coli O157:H7, Nature, 409, 529, 2001. 17. Xia, T., Zhao, G., and Jensen, R. A., Loss of allosteric control but retention of the bifunctional catalytic competence of a fusion protein formed by excision of 260 base pairs from the 3' terminus of pheA from Erwinia herbicola, Appl.Environ.Microbiol., 58, 2792, 1992. 18. Fischer, R. S., Zhao, G., and Jensen, R. A., Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis , J.Gen.Microbiol., 137, 1293, 1991. 19. Jimenez, N., Gonzalez-Candelas, F., and Silva, F. J. , Prephenate dehydratase from the aphid endosymbiont (Buchnera) displays changes in the regulatory domain that suggest its desensitization to inhibition by phenylalanine, J.Bacteriol., 182, 2967, 2000. 20. Gowrishankar, J. and Pittard, J., Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon, J.Bacteriol., 150, 1130, 1982. 21. Gavini, N. and Davidson, B. E., Regulation of pheA expression by the pheR product in Escherichia coli is mediated through attenuation of transcription, J.Biol.Chem., 266, 7750, 1991. 22. Xia, T., Zhao, G., and Jensen, R. A., The pheA/tyrA/aroF region from Erwinia herbicola: an emerging comparative basis for analysis of gene organization and regulation in enteric bacteria, J.Mol.Evol., 36, 107, 1993. 23. Baldwin, G. S., McKenzie, G. H., and Davidson, B. E. , The self-association of chorismate mutase/prephenate dehydratase from Escherichia coli K12, Arch.Biochem.Biophys., 211, 76, 1981. 24. Zhang, S., Pohnert, G., Kongsaeree, P., Wilson, D. B., Clardy, J., and Ganem, B., Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins, J.Biol.Chem., 273, 6248, 1998. 25. Stewart, J., Wilson, D. B., and Ganem, B., A genetically engineered monofunctional chorismate mutase, J. Am. Chem. Soc., 112, 4582, 1990. 26. Zhang, S., Wilson, D. B., and Ganem, B., Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain, Biochemistry, 39, 4722, 2000. 27. Xie, G., Brettin, T. S., Bonner, C. A., and Jensen, R. A., Mixed-function supraoperons that exhibit overall conservation, albeit shuffled gene organization, across wide intergenomic distances within eubacteria, Microb.Comp.Genomics, 4, 5, 1999. 28. Xie, G., Bonner, C. A., and Jensen, R. A., A probable mixed-function supraoperon in Pseudomonas exhibits gene organization features of both intergenomic conservation and gene shuffling, J.Mol.Evol., 49, 108, 1999. 29. Nelms, J., Edwards, R. M., Warwick, J., and Fotheringham, I., Novel mutations in 111 the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase, Appl.Environ.Microbiol., 58, 2592, 1992. ford, K. M., van Vliet, A. H., Whitehead, S., and Barrell, B. G., The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences, Nature, 403, 665, 2000. 30. Pohnert, G., Zhang, S., Husain, A., Wilson, D. B., and Ganem, B., Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein, Biochemistry, 38, 12212, 1999. 36. Tettelin, H., Saunders, N. J., Heidelberg, J., Jeffries, A. C., Nelson, K. E., Eisen, J. A., Ketchum, K. A., Hood, D. W., Peden, J. F., Dodson, R. J., Nelson, W. C., Gwinn, M. L., DeBoy, R., Peterson, J. D., Hickey, E. K., Haft, D. H., Salzberg, S. L., White, O., Fleischmann, R. D., Dougherty, B. A., Mason, T., Ciecko, A., Parksey, D. S., Blair, E., Cittone, H., Clark, E. B., Cotton, M. D., Utterback, T. R., Khouri, H., Qin, H., Vamathevan, J., Gill, J., Scarlato, V., Masignani, V., Pizza, M., Grandi, G., Sun, L., Smith, H. O., Fraser, C. M. , Moxon, E. R., Rappuoli, R., and Venter, J. C., Complete genome sequence of Neisseria meningitidis serogroup B strain MC58, Science, 287, 1809, 2000. 31. Nelson, K. E., Clayton, R. A., Gill, S. R., Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K. , Peterson, J. D., Nelson, W. C., Ketchum, K. A., McDonald, L., Utterback, T. R., Malek, J. A., Linher, K. D., Garrett, M. M., Stewart, A. M., Cotton, M. D., Pratt, M. S., Phillips, C. A., Richardson, D., Heidelberg, J., Sutton, G. G., Fleischmann, R. D., Eisen, J. A., and Fraser, C. M., Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima, Nature, 399, 323, 1999. 32. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A., and Merrick, J. M., Whole-genome random sequencing and assembly of Haemophilus influenzae Rd, Science, 269, 496, 1995. 33. Deckert, G., Warren, P. V., Gaasterland, T., Young, W. G., Lenox, A. L., Graham, D. E., Overbeek, R. , Snead, M. A., Keller, M., Aujay, M., Huber, R. , Feldman, R. A., Short, J. M., Olsen, G. J., and Swanson, R. V., The complete genome of the hyperthermophilic bacterium Aquifex aeolicus, Nature, 392, 353, 1998. 34. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., and Ishikawa, H., Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS, Nature, 407, 81, 2000. 35. Parkhill, J., Wren, B. W., Mungall, K., Ketley, J. M., Churcher, C., Basham, D., Chillingworth, T., Davies, R. M., Feltwell, T., Holroyd, S., Jagels, K., Karlyshev, A. V., Moule, S., Pallen, M. J., Penn, C. W., Quail, M. A., Rajandream, M. A., Ruther- 112 37. Parkhill, J., Achtman, M., James, K. D., Bentley, S. D., Churcher, C., Klee, S. R., Morelli, G., Basham, D., Brown, D., Chillingworth, T., Davies, R. M., Davis, P., Devlin, K., Feltwell, T., Hamlin, N., Holroyd, S., Jagels, K., Leather, S., Moule, S., Mungall, K., Quail, M. A., Rajandream, M. A., Rutherford, K. M., Simmonds, M., Skelton, J., Whitehead, S., Spratt, B. G., and Barrell, B. G., Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491, Nature, 404, 502, 2000. 38. Heidelberg, J. F., Eisen, J. A., Nelson, W. C., Clayton, R. A., Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K., Peterson, J. D., Umayam, L., Gill, S. R., Nelson, K. E., Read, T. D., Tettelin, H., Richardson, D., Ermolaeva, M. D., Vamathevan, J., Bass, S., Qin, H., Dragoi, I., Sellers, P., McDonald, L., Utterback, T., Fleishmann, R. D., Nierman, W. C., and White, O., DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae, Nature, 406, 477, 2000. 39. Simpson, A. J., Reinach, F. C., Arruda, P., Abreu, F. A., Acencio, M., Alvarenga, R., Alves, L. M. , Araya, J. E., Baia, G. S., Baptista, C. S., Barros, M. H., Bonaccorsi, E. D., Bordin, S., Bove, J. M., Briones, M. R., Bueno, M. R., Camargo, A. A., Camargo, L. E., Carraro, D. M., Carrer, H., Colauto, N. B., Colombo, C., Costa, F. F., Costa, M. C., Costa-Neto, C. M., Coutinho, L. L., Cristofani, M., Dias-Neto, E., Docena, C., El Dorry, H., Facincani, A. P., Ferreira, A. J., Ferreira, V. C., Ferro, J. A., Fraga, J. S., Franca, S. C., Franco, M. C., Frohme, M., Furlan, L. R., Garnier, M., Goldman, G. H., Goldman, M. H., Gomes, S. L., Gruber, A., Ho, P. L., Hoheisel, J. D., Junqueira, M. L., Kemper, E. L., Kitajima, J. P., and Marino, C. L., The genome sequence of the plant pathogen Xylella fastidiosa, Nature, 406, 151, 2000. 40. May, B. J. Zhang, Q., Li, L. L., Paustian, M. F., Whittam, T. S., and Kapur, V., Complete nucleotide sequence of an avian isolate of Pasteurella multocida, Proc.Natl.Acad.Sci.U.S.A., in press, 2001. 41. Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., WestbrockWadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K., Wu, Z., and Paulsen, I. T., Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen, Nature, 406, 959, 2000. 42. Hudson, G. S. and Davidson, B. E., Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12, J.Mol.Biol., 180, 1023, 1984. 43. Pittard, A. J. and Davidson, B. E., TyrR protein of Escherichia coli and its role as repressor and activator, Mol.Microbiol., 5, 1585, 1991. 44. Davidson, B. E. and Hudson, G. S., Chorismate mutase-prephenate dehydrogenase from Escherichia coli, Methods Enzymol., 142, 440, 1987. 45. Hudson, G. S., Wong, V., and Davidson, B. E., Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine, Biochemistry, 23, 6240, 1984. 46. Christendat, D. and Turnbull, J., Identification of active site residues of chorismate mutase-prephenate dehydrogenase from Escherichia coli, Biochemistry, 35, 4468, 1996. 47. Christopherson, R. I., Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogues of chorismate, Int.J.Biochem.Cell Biol., 29, 589, 1997. 48. Christendat, D. and Turnbull, J. L., Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli, Biochemistry, 38, 4782, 1999. 49. Turnbull, J. and Morrison, J. F., Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites, Biochemistry, 29, 10255, 1990. 50. Maruya, A., O’Connor, M. J., and Backman, K., Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product, J.Bacteriol., 169, 4852, 1987. 51. Turnbull, J., Morrison, J. F., and Cleland, W. W., Kinetic studies on chorismate mutase-prephenate dehydrogenase from Escherichia coli: models for the feedback inhibition of prephenate dehydrogenase by L-tyrosine, Biochemistry, 30, 7783, 1991. 52. Xie, G., Bonner, C. A., and Jensen, R. A., Cyclohexadienyl dehydrogenase from Pseudomonas stutzeri exemplifies a widespread type of tyrosine-pathway dehydrogenase in the TyrA protein family, Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol., 125, 65, 2000. 53. Xia, T., Zhao, G., Fischer, R. S., and Jensen, R. A., A monofunctional prephenate dehydrogenase created by cleavage of the 5' 109 bp of the tyrA gene from Erwinia herbicola, J.Gen.Microbiol., 138, 1309, 1992. 54. Klenk, H. P., Clayton, R. A., Tomb, J. F., White, O., Nelson, K. E., Ketchum, K. A., Dodson, R. J., Gwinn, M., Hickey, E. K., Peterson, J. D., Richardson, D. L., Kerlavage, A. R., Graham, D. E., Kyrpides, N. C., Fleischmann, R. D., Quackenbush, J., Lee, N. H., Sutton, G. G., Gill, S., Kirkness, E. F., Dougherty, B. A., McKenney, K., Adams, M. D., Loftus, B., and Venter, J. C., The complete genome sequence of the hyperthermophilic, 113 sulphate-reducing archaeon Archaeoglobus fulgidus, Nature, 390, 364, 1997. 55. Bolotin, A., Khazak, V., Stoynova, N., Ratmanova, K., Yomantas, Y., and Kozlov, Y., Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain, Microbiology, 141, 2219, 1995. 56. Huang, L., Nakatsukasa, M., and Nester, E., Regulation of aromatic amino acid biosynthesis in Bacillus subtilis 168. Purification, characterization, and subunit structure of the bifunctional enzyme 3-deoxy-Darabinoheptulosonate 7-phosphate synthetasechorismate mutase, J.Biol.Chem., 249, 4467, 1974. 57. Huang, L., Montoya, A. L., and Nester, E. W., Characterization of the functional activities of the subunits of 3-deoxy-D-arabinoheptulosonate 7phosphate synthetase-chorismate mutase from Bacillus subtilis 168, J.Biol.Chem., 249, 4473, 1974. 58. Llewellyn, D. J., Daday, A., and Smith, G. D., Evidence for an artificially evolved bifunctional 3-deoxy-D-arabinoheptulosonate7-phosphate synthase-chorismate mutase in Bacillus subtilis, J.Biol.Chem., 255, 2077, 1980. 59. Nakatsukasa, W. M. and Nester, E. W., Regulation of aromatic amino acid biosynthesis in Bacillus subtilis 168. I. Evidence for and characterization of a trifunctional enzyme complex, J.Biol.Chem., 247, 5972, 1972. 60. Nester, E. W., Jensen, R. A., and Nasser, D. S., Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus, J.Bacteriol., 97, 83, 1969. 61. Henner, D. and Yanofsky, C., Biosynthesis of aromatic amino acids, in Bacillus subtilis and other Gram-positive bacteria: Biochemistry, physiology, and molecular genetics, Sonenshein, A. L., Hoch, J. A., and Losick, R., Eds., ASM Press, Washington, D.C., 1993, 269. 62. Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S., and Horikoshi, K., Complete genome sequence of the alkaliphilic bacte- 114 rium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis, Nucleic Acids Res., 28, 4317, 2000. 63. White, O., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., Nelson, W. C., Richardson, D. L., Moffat, K. S., Qin, H., Jiang, L., Pamphile, W., Crosby, M., Shen, M., Vamathevan, J. J., Lam, P., McDonald, L., Utterback, T., Zalewski, C., Makarova, K. S., Aravind, L., Daly, M. J. , and Fraser, C. M., Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1, Science, 286, 1571, 1999. 64. Gray, J. V., Golinelli-Pimpaneau, B., and Knowles, J. R., Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli, Biochemistry, 29, 376, 1990. 65. Görisch, H., Chorismate mutase from Streptomyces aureofaciens, Methods Enzymol., 142, 463, 1987. 66. Xia, T., Song, J., Zhao, G., Aldrich, H., and Jensen, R. A., The aroQ-encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola, J.Bacteriol., 175, 4729, 1993. 67. Smith, D. R., Doucette-Stamm, L. A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., and Reeve, J. N., Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics, J.Bacteriol., 179, 7135, 1997. 68. Kawarabayasi, Y., Hino, Y., Horikawa, H., Yamazaki, S., Haikawa, Y., Jin-no, K., Takahashi, M., Sekine, M., Baba, S., Ankai, A., Kosugi, H., Hosoyama, A., Fukui, S., Nagai, Y., Nishijima, K., Nakazawa, H., Takamiya, M., Masuda, S., Funahashi, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., and Kikuchi, H., Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, DNA Res., 6, 83, 1999. 69. Ng, W. V., Kennedy, S. P., Mahairas, G. G., Berquist, B., Pan, M., Shukla, H. D., Lasky, S. R., Baliga, N. S., Thorsson, V., Sbrogna, J., Swartzell, S., Weir, D., Hall, J., Dahl, T. A., Welti, R., Goo, Y. A., Leithauser, B., Keller, K., Cruz, R., Danson, M. J., Hough, D. W., Maddocks, D. G., Jablonski, P. E., Krebs, M. P., Angevine, C. M., and Dale, H., From the cover: genome sequence of Halobacterium species NRC-1, Proc.Natl.Acad.Sci.U.S.A., 97, 12176, 2000. 70. Rajagopalan, J. S., Taylor, K. M., and Jaffe, E. K. , 13C NMR studies of the enzymeproduct complex of Bacillus subtilis chorismate mutase, Biochemistry, 32, 3965, 1993. 71. Lorence, J. H. and Nester, E. W., Multiple molecular forms of chorismate mutase in Bacillus subtillis, Biochemistry, 6, 1541, 1967. 72. Chen, X., Antson, A. A., Yang, M., Li, P., Baumann, C., Dodson, E. J., Dodson, G. G., and Gollnick, P., Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus, J.Mol.Biol., 289, 1003, 1999. 73. Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N. , Naruo, K., Okumura, S. , Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M., and Tabata, S., Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions, DNA Res., 3, 109, 1996. 74. Schmidheini, T., Sperisen, P., Paravicini, G., Hutter, R., and Braus, G. , A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae, J.Bacteriol., 171, 1245, 1989. 75. Schmidheini, T., Mosch, H. U., Evans, J. N., and Braus, G., Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution, Biochemistry, 29, 3660, 1990. 76. Krappmann, S., Pries, R., Gellissen, G., Hiller, M., and Braus, G. H., HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization, J. Bacteriol., 182, 4188, 2000. 77. Krappmann, S., Helmstaedt, K., Gerstberger, T., Eckert, S., Hoffmann, B., Hoppert, M., Schnappauf, G., and Braus, G. H., The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated chorismate mutase, J.Biol.Chem., 274, 22275, 1999. 78. Copley, S. D. and Knowles, J. R., The conformational equilibrium of chorismate in solution: implications for the mechanism of the non-enzymic and the enzyme-catalyzed rearrangement of chorismate to prephenate, J.Am.Chem.Soc., 109, 5008, 1987. 79. Addadi, L., Jaffe, E. K., and Knowles, J. R., Secondary tritium isotope effects as probes of the enzymic and nonenzymic conversion of chorismate to prephenate, Biochemistry, 22, 4494, 1983. 80. Copley, S. D. and Knowles, J. R., The uncatalyzed Claisen rearrangement of chorsimate to prephenate prefers a transition state of chairlike geometry, J.Am.Chem.Soc., 107, 5306, 1985. 81. Sogo, S. G., Widlanski, T. S., Hoare, J. H., Grimshaw, C. E., Berchtold, G. A., and Knowles, J. R., Stereochemistry of the rearrangement of chorismate to prephenate: Chorismate mutase involves a chair transition state, J.Am.Chem.Soc., 106, 2701, 1984. 82. Gajewski, J. J., Jurayj, J., Kimbrough, D. R., Gande, M. E., Ganem, B., and Carpenter, B. K., On the mechanism of rearrangement of chorismic acid and related compounds, J.Am.Chem.Soc., 109, 1170, 1987. 83. Gustin, D. P. and Hilvert, D., Chemoenzymatic synthesis of isotopically labeled chorismic acids, J.Org.Chem., 64, 4935, 1999. 84. Andrews, P. R., Smith, G. D., and Young, I. G., Transition-state stabilization and enzymic catalysis. Kinetic and molecular orbital 115 studies of the rearrangement of chorismate to prephenate, Biochemistry, 12, 3492, 1973. 85. Bartlett, P. A. and Johnson, C. R., An inhibitor of chorismate mutase resembling the transition-state conformation, J.Am.Chem.Soc., 107, 7792, 1985. 86. Chook, Y. M., Gray, J. V., Ke, H., and Lipscomb, W. N., The monofunctional chorismate mutase from Bacillus subtilis. Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction, J.Mol.Biol., 240, 476, 1994. 87. Chook, Y. M., Ke, H., and Lipscomb, W. N., Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog, Proc.Natl.Acad.Sci.U.S.A., 90, 8600, 1993. 88. Gray, J. V., Eren, D., and Knowles, J. R., Monofunctional chorismate mutase from Bacillus subtilis: kinetic and 13C NMR studies on the interactions of the enzyme with its ligands, Biochemistry, 29, 8872, 1990. 89. Gray, J. V. and Knowles, J. R., Monofunctional chorismate mutase from Bacillus subtilis: FTIR studies and the mechanism of action of the enzyme, Biochemistry, 33, 9953, 1994. 90. Cload, S. T., Liu, D. R., Pastor, R. M., and Schultz, P. G., Mutagenesis study of active site residues in chorismate mutase from Bacillus subtilis, J.Am.Chem.Soc., 118, 1787, 1996. 91. Lyne, P. D., Mulholland, A. J., and Richards, W. G., Insights into chorismate mutase catalysis from a combined QM/MM simulation of the enzyme reaction, J.Am.Chem.Soc., 117, 11345, 1995. 92. Wiest, O. and Houk, K. N., Stabilization of the transition state of chorismate-prephenate rearrangement: An ab initio study of enzyme and antibody catalysis, J.Am.Chem.Soc., 117, 11628, 1995. 93. Haynes, M. R., Stura, E. A., Hilvert, D., and Wilson, I. A., Routes to catalysis: structure of a catalytic antibody and comparison with its natural counterpart, Science, 263, 646, 1994. 116 94. Kast, P., Hartgerink, J. D., Asif-Ullah, M., and Hilvert, D., Electrostatic catalysis of the Claisen reaarrangement: Probing the role of Glu78 in Bacillus subtilis chorismate mutase by genetic selection , J.Am.Chem.Soc., 118, 3069, 1996. 95. Kast, P., Asif-Ullah, M., Jiang, N., and Hilvert, D., Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis, Proc.Natl.Acad.Sci.U.S.A., 93, 5043, 1996. 96. Kast, P., Grisostomi, C., Chen, I. A., Li, S., Krengel, U., Xue, Y., and Hilvert, D., A strategically positioned cation is crucial for efficient catalysis by chorismate mutase, J. Biol. Chem., 275, 36832, 2000. 97. Mattei, P., Kast, P., and Hilvert, D., Bacillus subtilis chorismate mutase is partially diffusion-controlled, Eur.J.Biochem., 261, 25, 1999. 98. Ladner, J. E., Reddy, P., Davis, A., Tordova, M., Howard, A. J., and Gilliland, G. L., The 1.30 Å resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer, Acta Crystallogr.D Biol. Crystallogr., 56, 673, 2000. 99. Gamper, M., Hilvert, D., and Kast, P., Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy, Biochemistry, 39, 14087, 2000. 100. Lee, A. Y., Karplus, P. M., Ganem, B., and Clardy, J., Atomic structure of the burried catalytic pocket of Escherichia coli chorismate mutase, J.Am.Chem.Soc., 117, 3627, 1995. 101. Turnbull, J., Cleland, W. W., and Morrison, J. F., pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli, Biochemistry, 30, 7777, 1991. 102. Guilford, W. J., Copley, S. D., and Knowles, J. R., On the mechanism of the chorismate mutase reaction, J.Am.Chem.Soc., 109, 5013, 1987. 103. Liu, D. R., Cload, S. T., Pastor, R. M., and Schultz, P. G., Analysis of active site residues in Escherichia coli chorismate mutase by site-directed mutagenesis, J.Am.Chem.Soc., 118, 1789, 1996. 104. Lee, A. Y., Stewart, J. D., Clardy, J., and Ganem, B., New insight into the catalytic mechanism of chorismate mutases from structural studies, Chem.Biol., 2, 195, 1995. 105. Lee, A. Y., Zhang, S., Kongsaeree, P., Clardy, J., Ganem, B., Erickson, J. W., and Xie, D., Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis, Biochemistry, 37, 9052, 1998. 106. Xue, Y. and Lipscomb, W. N., Location of the active site of allosteric chorismate mutase from Saccharomyces cerevisiae, and comments on the catalytic and regulatory mechanisms, Proc.Natl.Acad.Sci.U.S.A., 92, 10595, 1995. 107. Xue, Y. and Lipscomb, W. N., The crystallization and preliminary X-ray analysis of allosteric chorismate mutase, J.Mol.Biol., 241, 273, 1994. 108. Xue, Y., Lipscomb, W. N., Graf, R., Schnappauf, G., and Braus, G., The crystal structure of allosteric chorismate mutase at 2.2-Å resolution, Proc.Natl.Acad.Sci.U.S.A., 91, 10814, 1994. 109. Lin, S. L., Xu, D., Li, A., Rosen, M., Wolfson, H. J., and Nussinov, R., Investigation of the enzymatic mechanism of the yeast chorismate mutase by docking a transition state analog, J.Mol.Biol., 271, 838, 1997. 110. Sträter, N., Schnappauf, G., Braus, G., and Lipscomb, W. N., Mechanisms of catalysis and allosteric regulation of yeast chorismate mutase from crystal structures, Structure, 5, 1437, 1997. 111. Sträter, N., Hakansson, K., Schnappauf, G., Braus, G., and Lipscomb, W. N., Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state, Proc.Natl.Acad.Sci.U.S.A., 93, 3330, 1996. 112. Schnappauf, G., Strater, N., Lipscomb, W. N., and Braus, G. H., A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions, Proc.Natl.Acad.Sci.U.S.A., 94, 8491, 1997. 113. Ma, J., Zheng, X., Schnappauf, G., Braus, G., Karplus, M., and Lipscomb, W. N., Yeast chorismate mutase in the R state: simulations of the active site, Proc.Natl.Acad.Sci.U.S.A., 95, 14640, 1998. 114. Schnappauf, G., Krappmann, S., and Braus, G. H. , Tyrosine and tryptophan act through the same binding site at the dimer interface of yeast chorismate mutase, J.Biol.Chem., 273, 17012, 1998. 115. Schnappauf, G., Lipscomb, W. N., and Braus, G. H., Separation of inhibition and activation of the allosteric yeast chorismate mutase, Proc.Natl.Acad.Sci.U.S.A., 95, 2868, 1998. 116. Ramilo, C., Braus, G., and Evans, J. N., A tyrosine residue is involved in the allosteric binding of tryptophan to yeast chorismate mutase, Biochim.Biophys.Acta, 1203, 71, 1993. 117. Graf, R. and Braus, G. H., Modulation of the allosteric equilibrium of yeast chorismate mutase by variation of a single amino acid residue, J.Bacteriol., 177, 1645, 1995. 118. Ikeda, M., Ozaki, A., and Katsumata, R., Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli, Appl.Microbiol.Biotechnol., 39, 318, 1993. 119. Jackson, D. Y., Jacobs, J. W., Sugasawara, R., Reich, S. H., Bartlett, P. A., and Schultz, P. G., An antibody-catalyzed Claisen rearrangement, J.Am.Chem.Soc., 110, 4841, 1988. 120. MacBeath, G., Kast, P., and Hilvert, D., Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity, Protein Sci., 7, 325, 1998. 121. MacBeath, G., Kast, P., and Hilvert, D., Probing enzyme quaternary structure by combinatorial mutagenesis and selection, Protein Sci., 7, 1757, 1998. 122. MacBeath, G., Kast, P., and Hilvert, D., Redesigning enzyme topology by directed evolution, Science, 279, 1958, 1998. 117 123. Khanjin, N. A., Snyder, J. P., and Menger, F. M., Mechanism of chorismate mutase: Contribution of conformational restriction to catalysis in the Claisen rearrangement, J.Am.Chem.Soc., 121, 11831, 1999. 124. Marti, S., Andres, J., Moliner, V., Silla, E., Tunon, I., and Bertran, J., A QM/MM study of the conformational equilibria in the chorismate mutase active site. The role of the enzymatic deformation energy contribution, J.Phys.Chem.B., 104, 11308, 2000. 125. Hall, R. J., Hindle, S. A., Burton, N. A., and Hillier, I. H., Aspects of hybrid QM/ MM calculations: The treatment of the QM/ MM interface region and geometry optimization with an application to chorismate mutase, J.Comp.Chem., 21, 1433, 2000. 126. Gilchrist, D. G. and Connelly, J. A., Chorismate mutase from Mung Bean and Sorghum, Methods Enzymol., 142, [54], 1987. 127. Mobley, E. M., Kunkel, B. N., and Keith, B., Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana, Gene, 240, 115, 1999. 128. Eberhard, J., Raesecke, H. R., Schmid, J., and Amrhein, N., Cloning and expression in yeast of a higher plant chorismate mutase. Molecular cloning, sequencing of the cDNA and characterization of the Arabidopsis thaliana enzyme expressed in yeast, FEBS Lett., 334, 233, 1993. 129. Eberhard, J., Ehrler, T. T., Epple, P., Felix, G., Raesecke, H. R., Amrhein, N., and Schmid, J., Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties, Plant J., 10, 815, 1996. 130. Eberhard, J., Bischoff, M., Raesecke, H. R., Amrhein, N., and Schmid, J., Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase, Plant Mol.Biol., 31, 917, 1996. 131. Lambert, K. N., Allen, K. D., and Sussex, I. M. , Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica, Mol.Plant Microbe Interact., 12, 328, 1999. 118 132. Popeijus, M., Blok, V. C., Cardle, L., Bakker, E., Phillips, M. S., Helder, J., Smant, G., and Jones, J. T., Analysis of genes expressed in second stage juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida using the expressed sequence tag approach, Nematology, 2, 567, 2000. 133. Criado, L. M., Martin, J. F., and Gil, J. A., The pab gene of Streptomyces griseus, encoding p-aminobenzoic acid synthase, is located between genes possibly involved in candicidin biosynthesis, Gene, 126, 135, 1993. 134. Brown, M. P., Aidoo, K. A., and Vining, L. C., A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis, Microbiology, 142, 1345, 1996. 135. Blanc, V., Gil, P., Bamas-Jacques, N., Lorenzon, S., Zagorec, M., Schleuniger, J., Bisch, D., Blanche, F., Debussche, L., Crouzet, J., and Thibaut, D., Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylaminoL-phenylalanine precursor of pristinamycin I, Mol.Microbiol., 23, 191, 1997. 136. Teng, C., Ganem, B., Doktor, S. Z., Nichols, B. P., Bhagtnagar, R. K., and Vining, L. C., Total biosynthesis of 4-amino-4-deoxychorismic acid: a key intermediate in the biosynthesis of paminobenzoic acid and L-p-aminophenylalanine, J.Am.Chem.Soc., 107, 5008, 1985. 137. Pittard, A. J., Biosynthesis of the aromatic amino acids, in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1987, 368. 138. Zalkin, H. and Murphy, T., Utilization of ammonia for tryptophan synthesis, Biochem.Biophys.Res.Commun., 67, 1370, 1975. 139. Zalkin, H. and Smith, J. L., Enzymes utilizing glutamine as an amide donor, Adv.Enzymol.Relat.Areas.Mol.Biol., 72, 87, 1998. 140. Nichols, B. P., Evolution of Genes and Enzymes of Tryptophan Biosynthesis, in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1996, 2638. 141. Yanofsky, C. and Crawford, I. P., The tryptophan operon, in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1987, 1454. 142. Crawford, I. P., Evolution of a biosynthetic pathway: the tryptophan paradigm, Annu.Rev.Microbiol., 43, 567, 1989. 143. Oppenheim, D. S. and Yanofsky, C., Translational coupling during expression of the tryptophan operon of Escherichia coli, Genetics, 95, 785, 1980. 144. Yanofsky, C., Kelley, R. L., and Horn, V., Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe, J.Bacteriol., 158, 1018, 1984. 145. Yanofsky, C. and Horn, V., Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift, J.Bacteriol., 176, 6245, 1994. 146. Gunsalus, R. P. and Yanofsky, C., Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor, Proc.Natl.Acad.Sci.U.S.A., 77, 7117, 1980. 147. Storbakk, N., Fenton, C., Riise, H. M., Nilsen, I. W., and El-Gewely, M. R., In vivo interaction between mutated tryptophan repressors of Escherichia coli, J.Mol.Biol., 256, 889, 1996. 148. Ramesh, V., Syed, S. E., Frederick, R. O., Sutcliffe, M. J., Barnes, M., and Roberts, G. C., NMR studies of the mode of binding of corepressors and inducers to Escherichia coli trp repressor, Eur.J.Biochem., 235, 804 , 1996. 149. Kelley, R. L. and Yanofsky, C., Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA, Proc.Natl.Acad.Sci.U.S.A., 82, 483, 1985. superrepressor mutant AV77, J.Mol.Biol., 253, 266, 1995. 152. Czernik, P. J., Shin, D. S., and Hurlburt, B. K., Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli, J.Biol.Chem., 269, 27869, 1994. 153. Lawson, C. L. and Carey, J., Tandem binding in crystals of a trp repressor/operator halfsite complex, Nature, 366, 178, 1993. 154. Youderian, P. and Arvidson, D. N., Direct recognition of the trp operator by the trp holorepressor: a review, Gene, 150, 1, 1994. 155. Somerville, R., The Trp repressor, a ligandactivated regulatory protein, Prog.Nucleic Acid Res.Mol.Biol., 42, 1, 1992. 156. Roesser, J. R. and Yanofsky, C., The effects of leader peptide sequence and length on attenuation control of the trp operon of E.coli, Nucleic Acids Res., 19, 795, 1991. 157. Landick, R., Yanofsky, C., Choo, K., and Phung, L., Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression, J.Mol.Biol., 216, 25, 1990. 158. Landick, R., Carey, J., and Yanofsky, C., Translation activates the paused transcription complex and restores transcription of the trp operon leader region, Proc.Natl.Acad.Sci.U.S.A., 82, 4663, 1985. 159. Landick, R., Turnbough, C. L., and Yanofsky, C., Transcription attenuation, in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1996, 1263. 160. Yanofsky, C., Transcription attenuation: Once viewed as a novel regulatory strategy, J.Bacteriol., 182, 1, 2000. 150. Liu, Y. C. and Matthews, K. S., trp repressor mutations alter DNA complex stoichiometry, J.Biol.Chem., 269, 1692, 1994. 161. Arvidson, D. N., Arvidson, C. G., Lawson, C. L., Miner, J., Adams, C., and Youderian, P., The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae, Nucleic Acids Res., 22, 1821, 1994. 151. Reedstrom, R. J. and Royer, C. A., Evidence for coupling of folding and function in trp repressor: physical characterization of the 162. Miozzari, G. F. and Yanofsky, C., The regulatory region of the trp operon of Serratia marcescens, Nature, 276, 684, 1978. 119 163. Stroynowski, I. and Yanofsky, C., Transcript secondary structures regulate transcription termination at the attenuator of S. marcescens tryptophan operon, Nature, 298, 34, 1982. 164. Miozzari, G. F. and Yanofsky, C., Gene fusion during the evolution of the tryptophan operon in enterobacteriaceae, Nature, 277, 486, 1979. 165. Guerrero, C., Mateos, L. M., Malumbres, M., and Martin, J. F., Directed mutagenesis of a regulatory palindromic sequence upstream from the Brevibacterium lactofermentum tryptophan operon, Gene, 138, 35, 1994. 166. Sano, K. and Matsui, K., Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glutamicacid-producing bacterium, Gene, 53, 191, 1987. 167. Matsui, K., Miwa, K., and Sano, K., Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum, J.Bacteriol., 169, 5330, 1987. 168. Heery, D. M. and Dunican, L. K., Cloning of the trp gene cluster from a tryptophanhyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence, Appl.Environ.Microbiol., 59, 791, 1993. 169. Sato, S., Nakada, Y., Kanaya, S., and Tanaka, T., Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG, Biochim.Biophys.Acta, 950, 303, 1988. 170. Henner, D. J., Band, L., and Shimotsu, H., Nucleotide sequence of the Bacillus subtilis tryptophan operon, Gene, 34, 169, 1985. 171. Slock, J., Stahly, D. P., Han, C. Y., Six, E. W., and Crawford, I. P., An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of paraaminobenzoic acid, and the dihydropteroate synthase gene, J.Bacteriol., 172, 7211, 1990. 172. Shimotsu, H., Kuroda, M. I., Yanofsky, C., and Henner, D. J., Novel form of transcrip- 120 tion attenuation regulates expression the Bacillus subtilis tryptophan operon, J.Bacteriol., 166, 461, 1986. 173. Babitzke, P., Regulation of tryptophan biosynthesis: Trp-ing the TRAP or how Bacillus subtilis reinvented the wheel, Mol.Microbiol., 26 , 1, 1997. 174. Antson, A. A., Brzozowski, A. M., Dodson, E. J., Dauter, Z., Wilson, K. S., Kurecki, T., Otridge, J., and Gollnick, P., 11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis, J.Mol.Biol., 244, 1, 1994. 175. Antson, A. A., Otridge, J., Brzozowski, A. M., Dodson, E. J., Dodson, G. G., Wilson, K. S., Smith, T. M., Yang, M., Kurecki, T., and Gollnick, P., The structure of trp RNAbinding attenuation protein, Nature, 374, 693, 1995. 176. Gollnick, P., Ishino, S., Kuroda, M. I., Henner, D. J., and Yanofsky, C. , The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis, Proc.Natl.Acad.Sci.U.S.A., 87, 8726, 1990. 177. Babitzke, P., Gollnick, P., and Yanofsky, C., The mtrAB operon of Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis, J.Bacteriol., 174, 2059, 1992. 178. Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X., and Gollnick, P., Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA, Nature, 401, 235, 1999. 179. Muto, Y., Oubridge, C., and Nagai, K., RNA-binding proteins: TRAPping RNA bases, Curr.Biol., 10, 19, 2000. 180. Yang, M., Chen, X., Militello, K., Hoffman, R., Fernandez, B., Baumann, C., and Gollnick, P., Alanine-scanning mutagenesis of Bacillus subtilis trp RNAbinding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding, J.Mol.Biol., 270, 696, 1997. 181. Babitzke, P. and Yanofsky, C., Structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, J.Biol.Chem., 270, 12452, 1995. 182. Yakhnin, A. V., Trimble, J. J., Chiaro, C. R., and Babitzke, P., Effects of mutations in the L-tryptophan binding pocket of the trp RNA-binding attenuation protein of Bacillus subtilis, J.Biol.Chem., 275, 4519, 2000. 183. Baumann, C., Xirasagar, S., and Gollnick, P., The trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis binds to unstacked trp leader RNA, J.Biol.Chem., 272, 19863, 1997. 184. Babitzke, P., Bear, D. G., and Yanofsky, C., TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides, Proc.Natl.Acad.Sci.U.S.A., 92, 7916, 1995. 185. Babitzke, P., Yealy, J., and Campanelli, D., Interaction of the trp RNA-Binding attenuation protein (TRAP) of Bacillus subtilis with RNA: effects of the number of GAG repeats, the nucleotides separating adjacent repeats, and RNA secondary structure, J. Bacteriol., 178, 5159, 1996. ribosome binding site of Bacillus subtilis, J.Bacteriol., 179, 2582, 1997. 190. Yang, M., de Saizieu, A., van Loon, A. P., and Gollnick, P., Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP), J.Bacteriol., 177, 4272, 1995. 191. Lee, A. I., Sarsero, J. P., and Yanofsky, C., A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis, J.Bacteriol., 178, 6518, 1996. 192. Sarsero, J. P., Merino, E., and Yanofsky, C., A Bacillus subtilis operon containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis, Proc.Natl.Acad.Sci.U.S.A., 97, 2656, 2000. 193. Sarsero, J. P., Merino, E., and Yanofsky, C., A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport, J.Bacteriol., 182, 2329, 2000. 194. Rivas, M. V., Jarvis, E. D., and Rudner, R., The structure of the trpE, trpD and 5' trpC genes of Bacillus pumilus, Gene, 87, 71, 1990. 186. Elliott, M. B., Gottlieb, P. A., and Gollnick, P., The mechanism of RNA binding to TRAP: initiation and cooperative interactions, RNA, 7, 85, 2001. 195. Kuroda, M. I., Shimotsu, H., Henner, D. J., and Yanofsky, C., Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons, J.Bacteriol., 167, 792, 1986. 187. Du, H., Yakhnin, A. V., Dharmaraj, S., and Babitzke, P., trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon, J. Bacteriol., 182, 1819, 2000. 196. Hoffman, R. J. and Gollnick, P., The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis, J.Bacteriol., 177, 839, 1995. 188. Merino, E., Babitzke, P., and Yanofsky, C., trp RNA-binding attenuation protein (TRAP)trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon, J. Bacteriol., 177, 6362, 1995. 197. Raya, R., Bardowski, J., Andersen, P. S., Ehrlich, S. D., and Chopin, A., Multiple transcriptional control of the Lactococcus lactis trp operon, J.Bacteriol., 180, 3174, 1998. 189. Du, H., Tarpey, R., and Babitzke, P., The trp RNA-binding attenuation protein regulates TrpG synthesis by binding to the trpG 198. Bardowski, J., Ehrlich, S. D., and Chopin, A., Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis, J.Bacteriol., 174, 6563, 1992. 121 199. van de Guchte, M., Ehrlich, D. S., and Chopin, A., tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon, Mol.Microbiol., 29, 61, 1998. 200. Bae, Y. M., Holmgren, E., and Crawford, I. P., Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli, J.Bacteriol., 171, 3471, 1989. 201. Bae, Y. M. and Stauffer, G. V., Genetic analysis of the attenuator of the Rhizobium meliloti trpE(G) gene, J.Bacteriol., 173, 3382, 1991. 202. Bae, Y. M. and Crawford, I. P., The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition, J.Bacteriol., 172, 3318, 1990. 203. Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A., Mochizuki, Y., Nakayama, S., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada, M., and Tabata, S., Complete genome structure of the nitrogenfixing symbiotic bacterium Mesorhizobium loti, DNA Res., 7, 331, 2000. 204. De Troch, P., Dosselaere, F., Keijers, V., de Wilde, P., and Vanderleyden, J., Isolation and characterization of the Azospirillum brasilense trpE(G) gene, encoding anthranilate synthase, Curr.Microbiol., 34, 27, 1997. 205. Lin, C., Paradkar, A. S., and Vining, L. C., Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator, Microbiology, 144, 1971, 1998. 206. Hankins, C. N. and Mills, S. E., Anthranilate synthase-amidotransferase (combined). A novel from of anthranilate synthase from Euglena gracilis, J.Biol.Chem., 251, 7774, 1976. 207. Essar, D. W., Eberly, L., and Crawford, I. P., Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of 122 Pseudomonas putida trpE and trpGDC, J.Bacteriol., 172, 867, 1990. 208. Essar, D. W., Eberly, L., Han, C. Y., and Crawford, I. P., DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa, J.Bacteriol., 172, 853, 1990. 209. Shinomiya, T., Shiga, S., and Kageyama, M., Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. I. Localization of the pyocin R2 gene cluster between the trpCD and trpE genes, Mol.Gen.Genet., 189, 375, 1983. 210. Calhoun, D. H., Pierson, D. L., and Jensen, R. A., The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa, Mol.Gen.Genet., 121, 117, 1973. 211. Maurer, R. and Crawford, I. P., New regulatory mutation affecting some of the tryptophan genes in Pseudomonas putida, J.Bacteriol., 106, 331, 1971. 212. da Costa e Silva, O. and Kosuge, T., Molecular characterization and expression analysis of the anthranilate synthase gene of Pseudomonas syringae subsp. savastanoi, J.Bacteriol., 173, 463, 1991. 213. Ross, C. M. and Winkler, M. E., Regulation of tryptophan biosynthesis in Caulobacter crescentus, J.Bacteriol., 170, 769, 1988. 214. Wegman, J. and Crawford, I. P., Tryptophan synthetic pathway and its regulation in Chromobacterium violaceum, J.Bacteriol., 95, 2325, 1968. 215. Lai, C. Y., Baumann, P., and Moran, N., The endosymbiont (Buchnera sp.) of the aphid Diuraphis noxia contains plasmids consisting of trpEG and tandem repeats of trpEG pseudogenes, Appl.Environ.Microbiol., 62, 332, 1996. 216. Lai, C. Y., Baumann, L., and Baumann, P., Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids, Proc.Natl.Acad.Sci.U.S.A., 91, 3819, 1994. 217. Munson, M. A. and Baumann, P., Molecular cloning and nucleotide sequence of a putative trpDC(F)BA operon in Buchnera aphidicola (endosymbiont of the aphid Schizaphis graminum), J.Bacteriol., 175, 6426, 1993. 218. Wernegreen, J. J. and Moran, N. A., Decay of mutualistic potential in aphid endosymbionts through silencing of biosynthetic loci: Buchnera of Diuraphis, Proc.R.Soc.Lond.B.Biol.Sci., 267, 1423, 2000. 219. Van Ham, R. C. H. J., Martinez, T. D., Moya, A., and Latorre, A., Plasmid-encoded anthranilate synthase (TrpEG) in Buchnera aphidicola from aphids of the family Pemphigidae, Appl.Environ.Microbiol., 65, 117, 1999. 220. Baumann, L., Baumann, P., and Moran, N. A., The endosymbiont (Buchnera) of the aphid Diuraphis noxia contains all the genes of the tryptophan biosynthetic pathway, Curr.Microbiol., 37, 58, 1998. 221. Rouhbakhsh, D., Clark, M. A., Baumann, L., Moran, N. A., and Baumann, P., Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon, Mol.Phylogenet.Evol., 8, 167, 1997. 222. Dietrich, F. S., Mulligan, J., Hennessy, K., Yelton, M. A., Allen, E., Araujo, R., Aviles, E., Berno, A., Brennan, T., Carpenter, J., Chen, E., Cherry, J. M., Chung, E. , Duncan, M., Guzman, E. , Hartzell, G., HunickeSmith, S., Hyman, R. W., Kayser, A., Komp, C., Lashkari, D., Lew, H., Lin, D., Mosedale, D., Davis, R. W., et al., The nucleotide sequence of Saccharomyces cerevisiae chromosome V, Nature, 387, 78, 1997. 223. Zalkin, H., Paluh, J. L., van Cleemput, M., Moye, W. S., and Yanofsky, C., Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase, J.Biol.Chem., 259, 3985, 1984. 224. Dujon, B., Alexandraki, D., Andre, B., Ansorge, W., Baladron, V., Ballesta, J. P., Banrevi, A., Bolle, P. A., Bolotin-Fukuhara, M., and Bossier, P., Complete DNA sequence of yeast chromosome XI, Nature, 369, 371, 1994. 225. Hinnebusch, A. G., Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae, Microbiol.Rev., 52, 248, 1988. 226. Hollenbeck, J. J. and Oakley, M. G., GCN4 binds with high affinity to DNA sequences containing a single consensus half-site, Biochemistry, 39, 6380, 2000. 227. Braus, G. H., Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway, Microbiol.Rev., 55, 349, 1991. 228. Martens, J. A. and Brandl, C. J., GCN4p activation of the yeast TRP3 gene is enhanced by ABF1p and uses a suboptimal TATA element, J.Biol.Chem., 269, 15661, 1994. 229. Schechtman, M. G. and Yanofsky, C., Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli, J.Mol.Appl.Genet., 2, 83, 1983. 230. Nakai, R., Sen, K., Kurosawa, S., and Shibai, H., Cloning and sequencing analysis of Trp1 gene of Flammulina velutipes, FEMS Microbiol.Lett., 190, 51, 2000. 231. Kos, T., Kuijvenhoven, A., Hessing, H. G., Pouwels, P. H., and van den Hondel, C. A., Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms, Curr.Genet., 13, 137, 1988. 232. Caligiuri, M. G. and Bauerle, R., Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site, J.Biol.Chem., 266, 8328, 1991. 233. Graf, R., Mehmann, B., and Braus, G. H., Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae, J.Bacteriol., 175, 1061 , 1993. 233bis. Tang, X., Ezaki, S., Atomi, H., and Imanaka, T., Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan, Biochem.Biophys.Res.Commun., 281, 858, 2001. 234. Caligiuri, M. G. and Bauerle, R., Subunit communication in the anthranilate synthase complex from Salmonella typhimurium, Science, 252, 1845, 1991. 235. Tso, J. Y. and Zalkin, H., Chemical modifications of Serratia marcescens anthranilate synthase component I, J.Biol.Chem., 256, 9901, 1981. 123 236. Shiratsuchi, A. and Sato, S., Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis, J.Biochem.(Tokyo), 112, 714, 1992. 237. Morollo, A. A. and Bauerle, R., Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase, Proc.Natl.Acad.Sci.U.S.A., 90, 9983, 1993. 238. Bauerle, R., Hess, J., and French, S., Anthranilate synthase-anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium, Methods Enzymol., 142, 366, 1987. 239. Policastro, P. P., Au, K. G., Walsh, C. T., and Berchtold, G. A., trans-6-amino-5-[(1carboxyethenyl)oxy]-1,3-cyclo-hexadiene1-carboxylic acid: An intermediate in the biosynthesis of anthranilate from chorismate, J.Am.Chem.Soc., 106, 2443, 1984. 240. Teng, C. and Ganem, B., Shikimate-derived metabolites. 13. A key intermediate in teh biosynthesis of anthranilate from chorismate, J.Am.Chem.Soc., 106, 2463, 1984. 241. Walsh, C. T., Erion, M. D., Walts, A. E., Delany, J. J., and Berchtold, G. A., Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase, Biochemistry, 26, 4734, 1987. 242. Kozlowski, M. C., Tom, N. J., Seto, C. T., Sefler, A. M., and Bartlett, P. A., Chorismateutilizing enzymes isochoris-mate synthase, Anthranilate synthase, and p-Aminobenzoate synthase: mechanistic insight through inhibitor design, J.Am.Chem.Soc., 117, 2128, 1995. 243. Summerfield, A. E., Bauerle, R., and Grisham, C. M. , Magnetic resonance and kinetic studies of the partial complex and component I subunit forms of Salmonella typhimurium anthranilate synthase, J.Biol.Chem., 263, 18793, 1988. 244. Zalkin, H., The amidotransferases, Adv. Enzymol.Relat.Areas.Mol.Biol., 66, 203, 1993. 124 245. Morollo, A. A. and Eck, M.J., Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium, Nat.Struct.Biol. 8, 243, 1998. 246. Tso, J. Y., Bower, S. G., and Zalkin, H., Mechanism of inactivation of glutamine amidotransferases by the antitumor drug L(alpha S, 5S)-alpha-amino-3-chloro-4,5dihydro-5-isoxazoleacetic acid (AT-125), J.Biol.Chem., 255, 6734, 1980. 247. Kawamura, M., Keim, P. S., Goto, Y., Zalkin, H., and Heinrikson, R. L., Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis, J.Biol.Chem., 253, 4659, 1978. 248. Tso, J. Y., Hermodson, M. A., and Zalkin, H., Primary structure of Serratia marcescens anthranilate synthase component II, J.Biol.Chem., 255, 1451, 1980. 249. Paluh, J. L., Zalkin, H., Betsch, D., and Weith, H. L., Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis, J.Biol.Chem., 260, 1889, 1985. 250. Amuro, N., Paluh, J. L., and Zalkin, H., Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase, J.Biol.Chem., 260, 14844, 1985. 251. Bower, S. and Zalkin, H., Modification of Serratia marcescens anthranilate synthase with pyridoxal 5'-phosphate, Arch.Biochem.Biophys., 219, 121, 1982. 252. Goto, Y., Zalkin, H., Keim, P. S., and Heinrikson, R. L., Properties of anthranilate synthetase component II from Pseudomonas putida, J.Biol.Chem., 251, 941, 1976. 253. Knöchel, T., Ivens, A., Hester, G., Gonzalez, A., Bauerle, R., Wilmanns, M., Kirschner, K., and Jansonius, J. N., The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications, Proc.Natl.Acad.Sci.U.S.A., 96, 9479, 1999. 254. Tutino, M. L., Tosco, A., Marino, G., and Sannia, G., Expression of Sulfolobus solfataricus trpE and trpG genes in E. coli, Biochem.Biophys.Res.Commun., 230, 306, 1997. 255. Ozenberger, B. A., Brickman, T. J., and McIntosh, M. A., Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes, J.Bacteriol., 171, 775, 1989. 256. Spraggon, G., Kim, Co, Nguyen-Hun, X., Yee, M., Yanofsky, C., and Mills, S. E., The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan, Proc.Natl.Acad.Sci.U.S.A., 98, 6021, 2001. 257. Essar, D. W., Eberly, L., Hadero, A., and Crawford, I. P., Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications, J.Bacteriol., 172, 884, 1990. 258. Romling, U., Duchene, M., Essar, D. W., Galloway, D., Guidi-Rontani, C. , Hill, D., Lazdunski, A., Miller, R. V., Schleifer, K. H., and Smith, D. W., Localization of alg, opr, phn, pho, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO, J.Bacteriol., 174, 327, 1992. 259. Anjaiah, V., Koedam, N., Nowak-Thompson, B., Loper, J. E., Hofte, M., Tambong, J. T., and Cornelis, P., Involvement of phenazines and anthranilate in the antagonism of Pseudomonas aeruginosa PNA1 and Tn5 derivatives toward Fusarium spp. and Pythium spp., Mol.Plant Microbe Interact., 11, 847, 1998. 260. Mavrodi, D. V., Ksenzenko, V. N., Bonsall, R. F., Cook, R. J., Boronin, A. M., and Thomashow, L. S., A seven-gene locus for synthesis of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79, J.Bacteriol., 180, 2541, 1998. 261. Pierson, L. S., Gaffney, T., Lam, S., and Gong, F., Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30-84, FEMS Microbiol.Lett., 134, 299, 1995. 262. Thomashow, L. S. and Mavrodi, D. V. The genetics and regulation of antibiotics by PGPR, in Proceedings of the Fourth International Workshop on Plant Growth-Promoting Rhizobacteria, Ogoshi, A., Kobayashi, K., Homma, Y., Kodama, F., Kondo, N., and Akino, S., Eds., Japan-OECD Joint Workshop, Sapporo, 1997, 108. 263. Kutchan, T. M., Heterologous expression of alkaloid biosynthetic genes: a review, Gene, 179, 73, 1996. 264. Poulsen, C., Bongaerts, R. J., and Verpoorte, R., Purification and characterization of anthranilate synthase from Catharanthus roseus, Eur.J.Biochem., 212, 431, 1993. 265. Romero, R. M. and Roberts, M. F., Anthranilate synthase from Ailanthus altissima cell suspension cultures, Phytochemistry, 41, 402, 1996. 266. Niyogi, K. K. and Fink, G. R., Two anthranilate synthase genes in Arabidopsis: defenserelated regulation of the tryptophan pathway, Plant Cell, 4, 721, 1992. 267. Niyogi, K. K., Last, R. L., Fink, G. R., and Keith, B., Suppressors of trp1 fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase β subunit, Plant Cell, 5, 1011, 1993. 268. Bohlmann, J., Lins, T., Martin, W., and Eilert, U., Anthranilate synthase from Ruta graveolens. Duplicated AS a genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis, Plant Physiol., 111, 507, 1996. 269. Song, H. S., Brotherton, J. E., Gonzales, R. A., and Widholm, J. M., Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase, Plant Physiol., 117, 533, 1998. 270. Nichols, B. P., Seibold, A. M., and Doktor, S. Z., para-aminobenzoate synthesis from chorismate occurs in two steps, J.Biol.Chem., 264, 8597, 1989. 271. Ye, Z. Z., Liu, J., and Walsh, C. T., paminobenzoate biosynthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and en- 125 zyme X as an aminodeoxychorismate lyase, Proc.Natl.Acad.Sci.U.S.A., 87, 9391, 1990. 272. Green, J. M. and Nichols, B. P., pAminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC, J.Biol.Chem., 266, 12971, 1991. 273. Goncharoff, P. and Nichols, B. P., Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of paminobenzoate synthetase and anthranilate synthetase, J.Bacteriol., 159, 57, 1984. 274. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S. C., Bron, S., Brouillet, S., Bruschi, C. V., Caldwell, B., Capuano, V., Carter, N. M., Choi, S. K., Codani, J. J., Connerton, I. F., Danchin, A., et al., The complete genome sequence of the Gram-positive bacterium Bacillus subtilis, Nature, 390, 249, 1997. 275. Arhin, F. F. and Vining, L. C., Organization of the genes encoding p-aminobenzoic acid synthetase from Streptomyces lividans 1326, Gene, 126, 129, 1993. 276. Edman, J. C., Goldstein, A. L., and Erbe, J. G., p-Aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme, Yeast, 9, 669, 1993. 277. Anderson, K. S., Kati, W. M., Ye, Q. Y., Walsh, C. T., Benesi, A. J., and Johnson, K. A., Isolation and structure elucidation of the 4-amino-4-deoxychorismate intermediate in the PABA pathway, J.Am.Chem.Soc., 113, 3196, 1991. 278. Rayl, E. A., Green, J. M., and Nichols, B. P., Escherichia coli aminodeoxychorismate synthase: Analysis of pabB mutations affecting catalysis and subunit association, Biochim.Biophys.Acta, 1295, 81, 1996. sequences of Salmonella typhimurium and Klebsiella aerogenes pabB, Mol.Biol.Evol., 5, 531, 1988. 281. Arhin, F. F. and Vining, L. C., Cloning, nucleotide sequence and expression in Streptomyces lividans and Escherichia coli of pabB from Lactococcus lactis subsp. lactis NCDO 496, J.Gen.Microbiol., 139, 1785, 1993. 282. Kaplan, J. B. and Nichols, B. P., Nucleotide sequence of Escherichia coli pabA and its evolutionary relationship to trp(G)D, J.Mol.Biol., 168, 451, 1983. 283. Sawula, R. V. and Crawford, I. P., Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation, J.Bacteriol., 112, 797, 1972. 284. Sawula, R. V. and Crawford, I. P., Anthranilate synthetase of Acinetobacter calcoaceticus. Separation and partial characterization of subunits, J.Biol.Chem., 248, 3573 , 1973. 285. Kane, J. F., Holmes, W. M., and Jensen, R. A., Metabolic interlock: the dual role of a folate pathway gene as an extra-operonic gene of tryptophan biosynthesis, J.Biol.Chem., 247, 1587, 1972. 286. Kane, J. F., Regulation of a common amidotransferase subunit, J.Bacteriol., 132, 419, 1977. 287. Buvinger, W. E., Stone, L. C., and Heath, H. E., Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans, J.Bacteriol., 147, 62, 1981. 288. Tran, P. V., Bannor, T. A., Doktor, S. Z., and Nichols, B. P., Chromosomal organization and expression of Escherichia coli pabA, J.Bacteriol., 172, 397, 1990. 289. Tran, P. V. and Nichols, B. P., Expression of Escherichia coli pabA, J.Bacteriol., 173, 3680, 1991. 279. Viswanathan, V. K., Green, J. M., and Nichols, B. P., Kinetic characterization of 4amino 4-deoxychorismate synthase from Escherichia coli, J.Bacteriol., 177, 5918, 1995. 290. Kaplan, J. B., Merkel, W. K., and Nichols, B. P., Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens, J.Mol.Biol., 183, 327, 1985. 280. Goncharoff, P. and Nichols, B. P., Evolution of aminobenzoate synthases: nucleotide 291. Green, J. M., Merkel, W. K., and Nichols, B. P., Characterization and sequence of Es- 126 cherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme, J.Bacteriol., 174, 5317, 1992. 292. Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K. H., Yoshimura, T., and Esaki, N., Three-dimensional structure of 4-Amino-4-deoxychorismate lyase from Escherichia coli, J.Biochem.(Tokyo), 128, 29, 2000. 293. Jhee, K. H., Yoshimura, T., Miles, E. W., Takeda, S., Miyahara, I., Hirotsu, K., Soda, K., Kawata, Y., and Esaki, N., Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry, J.Biochem.(Tokyo), 128, 679, 2000. 294. Roux, B. and Walsh, C. T., p-Aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA, Biochemistry, 31, 6904, 1992. 295. Roux, B. and Walsh, C. T., p-Aminobenzoate synthesis in Escherichia coli: mutational analysis of three conserved amino acid residues of the amidotransferase PabA, Biochemistry, 32 , 3763, 1993. 296. Gil, J. A., Liras, P., Naharro, G., Villanueva, J. R., and Martin, J. F., Regulation by aromatic amino acids of the biosynthesis of candicidin by Streptomyces griseus, J.Gen.Microbiol., 118, 189, 1980. 297. Vining, L. C. and Stuttard, C., Chloramphenicol, in Genetics and biochemistry of antibiotic production, Vining, L. C. and Stuttard, C., Eds., Butterworth-Heinemann, Boston, 1994, 505. 298. Gil, J. A., Criado, L. M., Alegre, T., and Martin, J. F., Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains, FEMS Microbiol.Lett., 58, 15, 1990. 299. Gil, J. A. and Hopwood, D. A., Cloning and expression of a p-aminobenzoic acid synthetase gene of the candicidin-producing Streptomyces griseus, Gene, 25, 119, 1983. 300. Gil, J. A., Naharro, G., Villanueva, J. R., and Martin, J. F., Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus, J.Gen.Microbiol., 131, 1279, 1985. 301. Liu, J., Quinn, N., Berchtold, G. A., and Walsh, C. T., Overexpression, purification, and characterization of isochorismate synthase (EntC), the first enzyme involved in the biosynthesis of enterobactin from chorismate, Biochemistry, 29, 1417, 1990. 302. Müller, R., Breuer, M., Wagener, A., Schmidt, K., and Leistner, E., Bacterial production of transdihydroxycyclohexadiene carboxylates by metabolic pathway engineering, Microbiology, 142, 1005, 1996. 303. Crosa, J. H., Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria, Microbiol.Mol.Biol.Rev., 61, 319, 1997. 304. Tummuru, M. K., Brickman, T. J., and McIntosh, M. A., The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product. Evidence that EntA does not contribute to isochorismate synthase activity, J.Biol.Chem., 264, 20547, 1989. 305. Kwon, O., Hudspeth, M. E., and Meganathan, R., Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis, J.Bacteriol., 178, 3252, 1996. 306. Gehring, A. M., Mori, I., and Walsh, C. T., Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF, Biochemistry, 37, 2648, 1998. 307. Hantash, F. M. and Earhart, C. F., Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF, J.Bacteriol., 182, 1768, 2000. 308. Rusnak, F., Liu, J., Quinn, N., Berchtold, G. A., and Walsh, C. T., Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase, Biochemistry, 29, 1425, 1990. 309. Søballe, B. and Poole, R. K., Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management, Microbiology, 145, 1817, 1999. 127 310. Meganathan, R., Biosynthesis of the isoprenoid quinones menaquinone (vitamin K2) and ubiquinone (coenzyme Q), in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1996, 642. 311. Rowland, B. M. and Taber, H. W., Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3dihydroxybenzoate, J.Bacteriol., 178, 854, 1996. 312. Taber, H. W., Respiratory chains, in Bacillus subtilis and other Gram-positive bacteria: Biochemistry, physiology, and molecular genetics, Sonenshein, A. L., Hoch, J. A., and Losick, R., Eds., ASM Press, Washington, D.C., 1993, 199. 313. Weische, A., Johanni, M., and Leistner, E., Biosynthesis of o-succinylbenzoic acid. I: Cell free synthesis of o-succinylbenzoic acid from isochorismic acid in enzyme preparations from vitamin K producing bacteria, Arch.Biochem.Biophys., 256, 212, 1987. 314. Meganathan, R., Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms, Vitam.Horm., 61, 173, 2001. 315. Brickman, T. J., Ozenberger, B. A., and McIntosh, M. A., Regulation of divergent transcription from the iron-responsive fepBentC promoter-operator regions in Escherichia coli, J.Mol.Biol., 212, 669, 1990. 316. Wyckoff, E. E., Stoebner, J. A., Reed, K. E., and Payne, S. M., Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis, J.Bacteriol., 179, 7055, 1997. 317. Dahm, C., Muller, R., Schulte, G., Schmidt, K., and Leistner, E., The role of isochorismate hydroxymutase genes entC and menF in enterobactin and menaquinone biosynthesis in Escherichia coli, Biochim.Biophys.Acta, 1425, 377, 1998. 318. Daruwala, R., Bhattacharyya, D. K., Kwon, O., and Meganathan, R., Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new 128 isochorismate synthase from Escherichia coli, J.Bacteriol., 179 , 3133, 1997. 319. Kaiser, A. and Leistner, E., Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli, Arch.Biochem.Biophys., 276, 331, 1990. 320. Daruwala, R., Kwon, O., Meganathan, R., and Hudspeth, M. E., A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene, FEMS Microbiol.Lett., 140, 159, 1996. 321. Müller, R., Dahm, C., Schulte, G., and Leistner, E., An isochorismate hydroxymutase isogene in Escherichia coli, FEBS Lett., 378, 131, 1996. 322. Shestopalov, A. I., Bogachev, A. V., Murtazina, R. A., Viryasov, M. B., and Skulachev, V. P., Aeration-dependent changes in composition of the quinone pool in Escherichia coli. Evidence of post-transcriptional regulation of the quinone biosynthesis, FEBS Lett., 404, 272, 1997. 323. May, J. J., Wendrich, T. M., and Marahiel, M. A., The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoateglycine-threonine trimeric ester bacillibactin, J.Biol.Chem., 2000. 324. Rowland, B. M., Grossman, T. H., Osburne, M. S. , and Taber, H. W., Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes, Gene, 178, 119, 1996. 325. Rowland, B., Hill, K., Miller, P., Driscoll, J., and Taber, H., Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes, Gene, 167, 105, 1995. 326. Qin, X. and Taber, H. W., Transcriptional regulation of the Bacillus subtilis menp1 promoter, J.Bacteriol., 178, 705, 1996. 327. Hill, K. F., Mueller, J. P., and Taber, H. W., The Bacillus subtilis menCD promoter is responsive to extracellular pH, Arch.Microbiol., 153, 355, 1990. 328. Driscoll, J. R. and Taber, H. W., Sequence organization and regulation of the Bacillus subtilis menBE operon, J.Bacteriol., 174, 5063, 1992. in Vibrio vulnificus virulence, Infect.Immun., 64, 2834, 1996. 329. Barghouthi, S., Young, R., Olson, M. O., Arceneaux, J. E., Clem, L. W., and Byers, B. R., Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila, J.Bacteriol., 171, 1811, 1989. 338. De Voss, J. J., Rutter, K., Schroeder, B. G., and Barry, C. E., III, Iron acquisition and metabolism by mycobacteria, J. Bacteriol., 181, 4443, 1999. 330. Barghouthi, S., Payne, S. M., Arceneaux, J. E., and Byers, B. R., Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila, J.Bacteriol., 173, 5121, 1991. 331. Griffiths, G. L., Sigel, S. P., Payne, S. M., and Neilands, J. B., Vibriobactin, a siderophore from Vibrio cholerae, J.Biol.Chem., 259, 383, 1984. 332. Keating, T. A., Marshall, C. G., and Walsh, C. T., Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains, Biochemistry, 39, 15513, 2000. 333. Tindale, A. E., Mehrotra, M., Ottem, D., and Page, W. J., Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress, Microbiology, 146, 1617, 2000. 334. Serino, L., Reimmann, C., Visca, P., Beyeler, M., Chiesa, V. D., and Haas, D., Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa, J.Bacteriol., 179, 248, 1997. 335. Serino, L., Reimmann, C., Baur, H., Beyeler, M., Visca, P., and Haas, D., Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa, Mol.Gen.Genet., 249, 217, 1995. 336. Mercado-Blanco, J., Der Drift, K. M., Olsson, P. E., Thomas-Oates, J. E., van Loon, L. C., and Bakker, P. A., Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374, J.Bacteriol., 183, 1909, 2001. 337. Litwin, C. M., Rayback, T. W., and Skinner, J., Role of catechol siderophore synthesis 339. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., and Barrell, B. G., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence, Nature, 393, 537, 1998. 340. Quadri, L. E., Sello, J., Keating, T. A., Weinreb, P. H., and Walsh, C. T., Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin, Chem.Biol., 5, 631, 1998. 341. Rakin, A., Noelting, C., Schubert, S., and Heesemann, J., Common and specific characteristics of the high-pathogenicity island of Yersinia enterocolitica, Infect.Immun., 67, 5265, 1999. 342. Zamir, L. O., Devor, K. A., Jensen, R. A., Tiberio, R., Sauriol, F., and Mamer, O., Biosynthesis of isochorismate in Klebsiella pneumoniae: origin of O-2, Can.J.Microbiol., 37, 276, 1991. 343. Poulsen, C. and Verpoorte, R., Roles of chorismate mutase, isochorismate synthase and anthranilate synthase in plants, Phytochemistry, 30, 377, 1991. 344. Ledüc, C., Birgel, I., Müller, R., and Leistner, E., Isochorismate hydroxymutase from a cell-suspension culture of Gallium mollugo L., Planta, 202, 206, 1997. 345. van Tegelen, L. J., Moreno, P. R., Croes, A. F., Verpoorte, R., and Wullems, G. J., Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus, Plant Physiol., 119, 705, 1999. 346. Meng, H., Pullman, G. S., and Pater, G. F., Cloning of a plant isochorismate synthase 129 (Accenssion No. AF0078080) (PGR98-214), Plant Physiol., 118, 1536, 1998. 347. Palm, C. J., Federspiel, N. A., and Davis, R. W., DAtA: database of Arabidopsis thaliana annotation, Nucleic Acids Res., 28, 102, 2000. 348. Mauch, F., Mauch-Mani, B., Gaille, C., Kull, B., Haas, D., and Reimmann, C., Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase, Plant J., 25, 67, 2001. 349. Verberne, M. C., Verpoorte, R., Bol, J. F., Mercado-Blanco, J., and Linthorst, H. J., Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance, Nat.Biotechnol., 18, 779, 2000. 350. Verpoorte, R., Van der Heijden, R., and Memelink, J., Engineering the plant cell factory for secondary metabolite production, Transgen.Res., 9, 323, 2000. 351. Lawrence, J., Cox, G. B., and Gibson, F., Biosynthesis of ubiquinone in Escherichia coli K-12: biochemical and genetic characterization of a mutant unable to convert chorismate into 4-hydroxybenzoate, J.Bacteriol., 118, 41, 1974. 352. Siebert, M., Bechthold, A., Melzer, M., May, U., Berger, U., Schroder, G., Schroder, J., Severin, K., and Heide, L., Ubiquinone biosynthesis. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli, FEBS Lett., 307, 347, 1992. 353. Nichols, B. P. and Green, J. M., Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase, J.Bacteriol., 174, 5309, 1992. 354. Nishimura, K., Nakahigashi, K., and Inokuchi, H., Location of the ubiA gene on the physical map of Escherichia coli, J.Bacteriol., 174, 5762, 1992. 355. Wu, G., Williams, H. D., Gibson, F., and Poole, R. K., Mutants of Escherichia coli affected in respiration: the cloning and nucleotide sequence of ubiA, encoding the membrane-bound phydroxybenzoate:octaprenyltransferase, J.Gen.Microbiol., 139, 1795, 1993. 130 356. Siebert, M., Severin, K., and Heide, L., Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvatelyase, Microbiology, 140, 897, 1994. 357. Søballe, B. and Poole, R. K., Aerobic and anaerobic regulation of the ubiCA operon, encoding enzymes for the first two committed steps of ubiquinone biosynthesis in Escherichia coli, FEBS Lett., 414, 373, 1997. 358. Søballe, B. and Poole, R. K., Requirement for ubiquinone downstream of cytochrome(s) b in the oxygen-terminated respiratory chains of Escherichia coli K-12 revealed using a null mutant allele of ubiCA, Microbiology, 144, 361, 1998. 359. Hughes, M. A. and Williams, P. A., Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3, J.Bacteriol., 183, 1225, 2001. 360. Stover, C., Mayhew, M. P., Holden, M. J., Howard, A., and Gallagher, D. T., Crystallization and 1.1-Å diffraction of chorismate lyase from Escherichia coli, J.Struct.Biol., 129, 96, 2000. 361. Kaneda, T., Obata, H., and Tokumoto, M., Aromatization of 4-oxocyclohexanecarboxylic acid to 4-hydroxybenzoic acid by two distinctive desaturases from Corynebacterium cyclohexanicum. Properties of two desaturases, Eur.J.Biochem., 218, 997, 1993. 362. Lösher, R. and Heide, L., Biosynthesis of phydroxybenzoate from p-coumarate and pcoumaryl-coenzyme A in cell-free extracts of Lithospermum erythrorhizon cell cultures, Plant Physiol., 106, 271, 1994. 363. Sommer, S. and Heide, L., Expression of bacterial chorismate pyruvate-lyase in tobacco: evidence for the presence of chorismate in the plant cytosol, Plant Cell Physiol., 39, 1240, 1998. 364. Siebert, M., Sommer, S., Li, S. M., Wang, Z. X., Severin, K., and Heide, L., Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco, Plant Physiol., 112, 811, 1996. 365. Sommer, S., Kohle, A., Yazaki, K., Shimomura, K., Bechthold, A., and Heide, L., Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene, Plant Mol.Biol., 39, 683, 1999. 366. Reiners, J. J., Messenger, L. J., and Zalkin, H., Immunological cross-reactivity of Escherichia coli anthranilate synthetase, glutamate synthase, and other proteins, J.Biol.Chem., 253, 1226, 1978. 367. Gogarten, J. P. and Olendzenski, L., Orthologs, paralogs and genome comparisons, Curr.Opin.Genet.Dev., 9, 630, 1999. 368. Krappmann, S., Lipscomb, W. N., and Braus, G. H., Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae, Proc.Natl.Acad.Sci.U.S.A., 97, 13585, 2000. 369. Pittard, A. J., Biosynthesis of the aromatic amino acids, in Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ed., ASM Press, Washington, D.C., 1996, 458. 370. Khodursky, A. B., Peter, B. J., Cozzarelli, N. R. , Botstein, D., Brown, P. O., and Yanofsky, C., DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli, Proc.Natl.Acad.Sci.U.S.A., 97, 12170, 2000. 371. Baker, T. I. and Crawford, I. P., Anthranilate synthetase. Partial purification and some kinetic studies on the enzyme from Escherichia coli, J.Biol.Chem., 241, 5577, 1966. 372. Dopheide, T. A., Crewther, P., and Davidson, B. E., Chorismate mutaseprephenate dehydratase from Escherichia coli K-12. II. Kinetic properties , J.Biol.Chem., 247, 4447, 1972. 373. Kunzler, M., Paravicini, G., Egli, C. M., Irniger, S., and Braus, G. H., Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxyD-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae, Gene, 113, 67, 1992. 374. Paravicini, G., Schmidheini, T., and Braus, G., Purification and properties of the 3-deoxyD-arabino-heptulosonate-7-phosphate synthase (phenylalanine-inhibitable) of Saccharomyces cerevisiae, Eur.J.Biochem., 186, 361, 1989. 375. Schnappauf, G., Hartmann, M., Kunzler, M., and Braus, G. H., The two 3-deoxy-Darabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition, Arch.Microbiol., 169, 517, 1998. 376. Teshiba, S., Furter, R., Niederberger, P., Braus, G., Paravicini, G., and Hutter, R., Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation, Mol.Gen.Genet., 205, 353, 1986. 377. Kunzler, M., Springer, C., and Braus, G. H., Activation and repression of the yeast ARO3 gene by global transcription factors, Mol.Microbiol., 15, 167, 1995. 131