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The turnover product of the committed step of menaquinone biosynthesis was isolated and determined to be (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate. Structural determination of this key intermediate represents a critical step to complete elucidation of the biosynthetic pathway.

Chorismic acid is the key branch point intermediate in the biosynthesis of aromatic amino acids in microorganisms and plants (Scheme 1.1a) [1]. In the branch that leads to the production of tyrosine and phenylalanine, chorismate mutase (CM, chorismate-pyruvate mutase, EC 5.4.99.5) is a key enzyme that catalyzes the isomerization of chorismate to prephenate (Scheme 1.1b) with a rate enhancement of about 10–10-fold. This reaction is one of few pericyclic processes in biology and provides a rare opportunity for understanding how Nature promotes such unusual transformations. The biological importance of the conversion from chorismate to prephenate and the synthetic value of the Claisen rearrangement have led to extensive experimental investigations [2–43]. In addition, the reaction catalyzed by chorismate mutase is a paradigm for the study of enzyme mechanism and has been a subject of extensive computational investigations [44, 47–83]. One of the main reasons for the current focus on the mechanism of this enzyme is the fact that the reaction is a straightforward unimolecular rearrangement of the substrate with no chemical transformations in the enzyme or the solvent during the reaction. This eliminates many of the problems that arise for other cases and may help to settle some of the long-standing issues concerning the origin of the catalysis [84]. Experimental results for the CM-catalyzed and uncatalyzed reaction, as well as structural information for chorismate mutase, have been extensively discussed in two previous reviews [2, 3]. There has been a rapid growth of literature in computational studies of chorismate mutase in the last few years. In this chapter, we shall begin by summarizing some key experimental data related to the Claisen rearrangement along with existing structural information for chorismate mutase. We will then review the results of computational studies of chorismate mutase and discuss different proposals that have been suggested for the mechanism of the CM-catalyzed reaction.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised patients and those with cystic fibrosis (CF). This gram-negative bacterium uses multiple cell-to-cell signals to control numerous cellular functions and virulence. One of these signals is 2-heptyl-3-hydroxy-4-quinolone, which is referred to as the Pseudomonas quinolone signal (PQS). This signal functions as a coinducer for a transcriptional regulator (PqsR) to positively control multiple virulence genes and its own synthesis. PQS production is required for virulence in multiple models of infection, and it has been shown to be produced in the lungs of CF patients infected by P. aeruginosa . One of the precursor compounds from which PQS is synthesized is the metabolite anthranilate. This compound can be derived from the conversion of chorismate to anthranilate by an anthranilate synthase or through the degradation of tryptophan via the anthranilate branch of the kynurenine pathway. In this study, we present data which help to define the kynurenine pathway in P. aeruginosa and show that the kynurenine pathway serves as a critical source of anthranilate for PQS synthesis. We also show that the kyn pathway genes are induced during growth with tryptophan and that they are autoregulated by kynurenine. This study provides solid foundations for the understanding of how P. aeruginosa produces the anthranilate that serves as a precursor to PQS and other 4-quinolones.

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.

MbtI (rv2386c) from Mycobacterium tuberculosis catalyzes the initial transformation in mycobactin biosynthesis by converting chorismate to salicylate. We report here the structure of MbtI at 2.5 A resolution and demonstrate that isochorismate is a kinetically competent intermediate in the synthesis of salicylate from chorismate. At pH values below 7.5 isochorismate is the dominant product while above this pH value the enzyme converts chorismate to salicylate without the accumulation of isochorismate in solution. The salicylate and isochorismate synthase activities of MbtI are Mg2+-dependent, and in the absence of Mg2+ MbtI has a promiscuous chorismate mutase activity similar to that of the isochorismate pyruvate lyase, PchB, from Pseudomonas aeruginosa. MbtI is part of a larger family of chorismate-binding enzymes descended from a common ancestor (the MST family), that includes the isochorismate synthases and anthranilate synthases. The lack of active site residues unique to pyruvate eliminating members of this family, combined with the observed chorismate mutase activity, suggests that MbtI may exploit a sigmatropic pyruvate elimination mechanism similar to that proposed for PchB. Using a combination of structural, kinetic, and sequence based studies we propose a mechanism for MbtI applicable to all members of the MST enzyme family.

Menaquinone biosynthesis is initiated by the conversion of chorismate to isochorismate, a reaction that is catalyzed by the menaquinone-specific isochorismate synthase, MenF. The catalytic mechanism of MenF has been probed using a combination of structural and biochemical studies, including the 2.5 A structure of the enzyme, and Lys190 has been identified as the base that activates water for nucleophilic attack at the chorismate C2 carbon. MenF is a member of a larger family of Mg2+ dependent chorismate binding enzymes catalyzing distinct chorismate transformations. The studies reported here extend the mechanism recently proposed for this enzyme family by He et al.: He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378−85.

Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K(eq) = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K(m) of 41.5 mum and k(cat) = 38.7 min(-1) for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg(2+), or redox and that it is remarkably active at 4 degrees C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

Molecular dynamics (MD) simulations of Thermus thermophilus chorismate mutase substrate complex (TtCM x S) have been carried out at 298 K, 333 K, and the temperature of optimum activity: 343 K. The enzyme exists as trimeric subunits with active sites shared between two neighboring subunits. Two features distinguish intersubunit linkages of the thermophilic and mesophilic enzyme Bacillus subtilis chorismate mutase substrate complex (BsCM x S): (i) electrostatic interactions by intersubunit ion pairs (Arg3-Glu40*/41, Arg76-Glu51* and Arg69*-Asp101, residues labeled with an asterisk are from the neighboring subunit) in the TtCM x S are not present in the structure of the BsCM x S; and (ii) replacement of polar residues with short and nonpolar residues in the interstices of the TtCM x S tighten the intersubunit hydrophobic interactions compared to BsCM x S. Concerning the active site, electrostatic interactions of the critically placed Arg6 and Arg63* with the two carboxylates of chorismate place the latter in a reactive conformation to spontaneously undergo a Claisen rearrangement. The optimum geometry at the active site has the CZ atoms of the two arginines 11 A apart. With a decrease in temperature, Arg63* moves toward Arg6 and the average conformation structure of chorismate moves further away from the reactive ground state conformation. This movement is due to the decrease in distance separating the electrostatic (in the main) and hydrophobic interacting pairs holding the two subunits together.

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.

pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with Km values for glutamine and chorismate of 600 and 1.5 μM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (Ki values of 10 and 1 μM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated at C2 as a substrate. Monitoring the reaction by 2H NMR spectroscopy revealed that the label is quantitatively transferred from C2 to C9, producing stoichiometric amounts of [3-2H]pyruvate as product. Moreover, the deuterium kinetic isotope effect of 2.34 +/- 0.08 on kcat indicates that C-H cleavage is significantly rate limiting. Consistent with these data, hybrid density functional theory (HDFT) calculations at the Becke3LYP/DZ+(2d,p) level of theory predict a concerted but highly asynchronous pericyclic transition structure, in which carbon-oxygen bond cleavage is more advanced than hydrogen atom transfer from C2 to C9; the calculated 2H isotope effect of 2.22 at C2 is in excellent accord with the experimental value. Together, these findings indicate that IPL should be added to the small set of proteins that are known to catalyze pericyclic reactions. They also raise the possibility that enzymes, such as chorismate pyruvate lyase, salicylate synthase, 4-amino-4-deoxychorismate lyase, and anthranilate synthase, which accelerate formally similar reaction steps, may also exploit pericyclic mechanisms.

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1 H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K m for chorismate of 4.2 μM and a k cat of 8 min −1 . The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1 H NMR spectroscopy.

Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase.

The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.

Reinvestigation of the reaction between (–)-chorismic acid and diazomethane in diethyl ether on a larger scale has shown that the previously reported single pyrazoline based product is accompanied by a stereoisomer that result from the addition of diazomethane to the more hindered α-face of the chorismate 1,2-double bond (α:β addition ca. 1:6). Thermolysis of the two cycloadducts at 80 °C afforded a pair of cyclopropane derivatives with stereochemistry that could not be confidently assigned using data from coupling constants alone. NOE data allowed a more confident assignment of the stereochemistry of the two cyclopropanes; the β-cyclopropyl derivative was saponified and hydrolysed to yield a bicyclo[4.1.0]hept-2-ene-1-carboxylic acid derivative that was unequivocally shown to possess (1S,4R,5R,6R)-stereochemistry by an X-ray crystallographic study.

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5′ untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.

Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.

PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5′-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5′-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH–PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.

Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent. Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source. Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.

Standard free energies for formation of ground-state reactive conformers (DeltaGN degrees ) and transition states (DeltaG) in the conversion of chorismate to prephenate in water, B. subtilis mutase, E. coli mutase, and their mutants, as well as a catalytic antibody, are related by DeltaG = DeltaGN degrees + 16 kcal/mol. Thus, the differences in the rate constants for the water reaction and catalysts reactions reside in the mole fraction of substrate present as reactive conformers (NACs). These results, and knowledge of the importance of transition state stabilization in other cases, suggest a proposal that enzymes utilize both NAC and transition state stabilization in the mix required for the most efficient catalysis.

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus , is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1- 13 C]mannose and intact incorporation of d -[1,2- 13 C 2 ]glucose into the 6-deoxy-5-keto- d -arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l -fucose, via a 4-keto-6-deoxy- d -mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d -[1,2- 13 C 2 ]glucose in a manner consistent with formation of myo -inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo -inosamine-2. Incorporation of [ carboxy- 13 C]-4-hydroxybenzoic acid and intact incorporation of [2,3- 13 C 2 ]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3- 13 C]tyrosine, [3- 13 C]phenylalanine, or [ carboxy- 13 C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N -(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new antibiotics which target the ribosomal peptidyl transferase activity.

Solvent effects on the rate of the Claisen rearrangement of chorismate to prephenate have been examined in water and methanol. The preequilibrium free-energy differences between diaxial and diequatorial conformers of chorismate, which had previously been implicated as the sole basis for the observed 100-fold rate increase in water over methanol, have been reframed using the near attack conformation (NAC) concept of Bruice and co-workers. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively. As a consequence, the conversion of non-NACs to NACs provides no free-energy contributions to the overall relative reaction rates in water versus methanol. Free-energy perturbation calculations yielded differences in free energies of activation for the two polar protic solvents and the gas phase. The rate enhancement in water over the gas phase arises from preferential hydration of the transition state (TS) relative to the reactants via increased hydrogen bonding and long-range electrostatic interactions, which accompany bringing the two negatively charged carboxylates into closer proximity. More specifically, there is an increase of 1.3 and 0.6 hydrogen bonds to the carboxylate groups and the ether oxygen, respectively, in going from the reactant to the TS in water. In methanol, the corresponding changes in hydrogen bonding with first shell solvent molecules are small; the rate enhancement arises primarily from the enhanced long-range interactions with solvent molecules. Thus, the reaction occurs faster in water than in methanol due to greater stabilization of the TS in water by specific interactions with first shell solvent molecules.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.

A double-stranded (ds)RNA, designated as M2, is associated with hypovirulence, conversion of the quinic acid pathway from inducible to constitutive and downregulation of the shikimic acid pathway in the Rhizoctonia solani culture Rhs 1A1. In this study, we report that in the virulent, M2-lacking isolate Rhs 1AP, which is isogenic to Rhs 1A1, quinic acid reduces virulence dramatically and induces synthesis of an M2-encoded polypeptide and its respective mRNA. The full-length sense strand of M2 is detected in untreated Rhs 1AP only after a second 30-cycle amplification, using nested primers. Quinate-induced Rhs 1AP contains low concentrations of both full-length sense and complementary strand of M2. The quinic acid-induced hypovirulence in Rhs 1AP cannot be overturned by the end-product of the shikimic acid pathway, chorismic acid, which enhances the virulence of Rhs 1AP dramatically when used alone. In addition to its apparent applications, this study confirms the strong association between the M2 dsRNA and hypovirulence in R. solani.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.

Salicylic acid (SA) mediates plant defences against pathogens, accumulating in both infected and distal leaves in response to pathogen attack. Pathogenesis-related gene expression and the synthesis of defensive compounds associated with both local and systemic acquired resistance (LAR and SAR) in plants require SA. In Arabidopsis, exogenous application of SA suffices to establish SAR, resulting in enhanced resistance to a variety of pathogens. However, despite its importance in plant defence against pathogens, SA biosynthesis is not well defined. Previous work has suggested that plants synthesize SA from phenylalanine; however, SA could still be produced when this pathway was inhibited, and the specific activity of radiolabelled SA in feeding experiments was often lower than expected. Some bacteria such as Pseudomonas aeruginosa synthesize SA using isochorismate synthase (ICS) and pyruvate lyase. Here we show, by cloning and characterizing an Arabidopsis defence-related gene (SID2) defined by mutation, that SA is synthesized from chorismate by means of ICS, and that SA made by this pathway is required for LAR and SAR responses.

Background Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. Results The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. Conclusions Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously.

Shikimate kinase (EC2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi‖1500322 inMethanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC2.7.1.39) of the GHMP-kinase superfamily. The latter functionality inM. jannaschiiis assigned to another gene (gi‖1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed inEscherichia coliand purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases:Km,shikimate= 414 ± 33 μM,Km,ATP= 48 ± 4 μM, andkcat= 57 ± 2 s−1for the predicted shikimate kinase andKm,homoserine= 188 ± 37 μM,Km,ATP= 101 ± 7 μM, andkcat= 28 ± 1 s−1for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq)+ammonia(aq)=anthranilate(aq)+pyruvate(aq)+H 2 O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq)+ammonia(aq)=ADIC(aq)+H 2 O(l) and ADIC(aq)=anthranilate(aq)+pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate 2− (aq)+NH 4 + (aq)=anthranilate − (aq)+pyruvate − (aq)+H + (aq)+H 2 O(l), Δ r H m o =−(116.3±5.4) kJ mol −1 . For the reaction: chorismate 2− (aq)+NH 4 + (aq)=ADIC − (aq)+H 2 O(l), K =(20.3±4.5) and Δ r H m o =(7.5±0.6) kJ mol −1 . Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes Δ r G′ m o under approximately physiological conditions are given.

Reaction of (−)-methyl shikimate ((−) with 2-acetoxyisobutyryl bromide afforded (+)-methyl (3R,4S,5R)-3-bromo-4-acetoxy-5-hydroxy-1-cyclohexene-1-carboxylate ((+). Transesterification of this bromoacetate with NaOCH 3 led quantitatively to the corresponding epoxyol, (+)-methyl 3,4-anhydroshikimate. Payne rearrangement of this trans-epoxyol produced (−)-methyl (3S,4S,5R)-3-hydroxy-4,5-epoxy-1-cyclohexene-1-carboxylate ((−), which has previously been converted into (−)-chorismic acid

Klebsiella pneumoniae 62-1, a triple mutant impaired in aromatic amino acid biosynthesis (Phe-, Tyr-, Trp-), excretes chorismic acid into the culture broth. When transformed with plasmids harbouring Escherichia coli genes entC or menF the mutant excretes a mixture of both chorismic and isochorismic acid indicating that not only entC but also menF encodes an isochorismate hydroxymutase (isochorismate synthase, EC 5.4.99.6) enzyme. These enzymes catalyze the first step in enterobactin or menaquinone biosynthesis, respectively. Although both gene products (EntC and MenF) catalyze the same reaction, they play distinct roles in the biosynthesis of menaquinone (MK8) and enterobactin. An E. coli mutant (PBB7) with an intact menF but a disrupted entC produced menaquinone (MK8) but no enterobactin, whereas a mutant (PBB9) with an intact entC but a disrupted menF produced enterobactin and only a trace of menaquinone (MK8). When both menF and entC were disrupted (mutant PBB8) neither menaquinone (MK8) nor enterobactin was detectable. Our previous assumption that entC is responsible for both menaquinone and enterobactin biosynthesis is inconsistent with these mutant studies and has to be revised. The presence in the promoter region of menF of a putative cAMP receptor protein binding site indicates that menF is regulated differently from entC. The menF gene was overexpressed as a fusion gene and its product (6xHis-tagged MenF) isolated. The enzyme catalyzed the formation of isochorismic from chorismic acid and as opposed to a previous publication also the reverse reaction. The enzyme was characterized and its kinetic data determined.

The structure of the complex of the chorismate mutase from the yeast Saccharomyces cerevisiae with a transition state analog is constructed using a suite of docking tools. The construction finds the best location for the active site in the enzyme, and the best orientation of the analog compound in the active site. The resulting complex shows extensive salt links and hydrogen bonds between the enzyme and the compound, including those mediated by water molecules. A network of polar interactions between amino acid residues is found to solidify the active site of the enzyme. The enzymatic mechanism suggested for a bacterial chorismate mutase, that the active site is by design capable of selecting an active conformer of the substrate, and of stabilizing the transition state, is apparently intact in the yeast enzyme. No direct evidence is found to support an alternative mechanism which involves specific catalytic groups, although the possibility is not eliminated. This finding reinforces the notion of a function being evolutionarily conserved via a common mechanism, rather than via sequential or structural homology.

A novel technique for computing free energy profiles in enzymatic reactions using the multiple steering molecular dynamics approach in the context of an efficient QM-MM density functional scheme is presented. The conversion reaction of chorismate to prephenate catalyzed by the Bacillus subtilis enzyme chorismate mutase has been chosen as an illustrative example.

We extend the Effective Fragment Molecular Orbital (EFMO) method to the frozen domain approach where only the geometry of an active part is optimized, while the many-body polarization effects are considered for the whole system. The new approach efficiently mapped out the entire reaction path of chorismate mutase in less than four days using 80 cores on 20 nodes, where the whole system containing 2398 atoms is treated in the ab initio fashion without using any force fields. The reaction path is constructed automatically with the only assumption of defining the reaction coordinate a priori. We determine the reaction barrier of chorismate mutase to be $18.3\pm 3.5$ kcal mol$^{-1}$ for MP2/cc-pVDZ and $19.3\pm 3.6$ for MP2/cc-pVTZ in an ONIOM approach using EFMO-RHF/6-31G(d) for the high and low layers, respectively., SI not attached

A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also be redefined.

Mutagenesis of Streptomyces venezuelae ISP5230 and selection for p-aminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants (VS519 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQ102 or pDQ103, which carried a pab-complementing fragment from S. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519. The cloned S. venezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHI-SacI fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N- and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. venezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 μg ml-1 and blocked Cm production.

Publisher Summary A number of attenuated mutants of Salmonella are able to interact with the lymphoid tissues in Peyer's patches, but not able to cause systemic disease. Some of these mutants are effective as live vaccines (i.e., able to protect against infection with the virulent Salmonella parent) and are candidates for use as carriers for other virulence determinants. Different mutants have been employed for this purpose, including galE mutants, which lack the enzyme uridine diphosphate (UDP)-galactose-4-epimerase, and aroA mutants, which have specific nonreverting deletions in the common aromatic biosynthetic pathway leading to chorismic acid. The number of reported uses of this system of antigen delivery for the development of both humoral and cell-mediated immunity increases daily.

The active site of the allosteric chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) was located by comparison with the mutase domain (ECM) of chorismate mutase/prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] (the P protein) from Escherichia coli. Active site domains of these two enzymes show very similar four-helix bundles, each of 94 residues which superimpose with a rms deviation of 1.06 A. Of the seven active site residues, four are conserved: the two arginines, which bind to the inhibitor's two carboxylates; the lysine, which binds to the ether oxygen; and the glutamate, which binds to the inhibitor's hydroxyl group in ECM and presumably in YCM. The other three residues in YCM (ECM) are Thr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88). This Glu-246, modeled close to the ether oxygen of chorismate in YCM, may function as a polarizing or ionizable group, which provides another facet to the catalytic mechanism.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti-stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6-fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406).

Publisher Summary This chapter provides an overview of the chloramphenicol. Chloramphenicol is an N-dichloroacyl phenylpropanoid metabolite produced by Streptomyces venezuelae and several other actinomycetes. Most of the work on chloramphenicol biosynthesis and on the physiology of its production in culture has been done with S. venezuelae 13s, a strain chosen by screening cultures initiated from single spores of a soil isolate for superior chloramphenicol production. There is adequate evidence from isotopic labeling experiments that the phenylpropanoid moiety of chloramphenicol is derived from intermediary metabolism via shikimic acid. The conclusion that chorismic acid is the shikimate pathway branch-point at which intermediates are diverted from the biosynthesis of primary aromatic metabolites into secondary metabolism is based on the experiments with cell extracts. The relationship between biomass accumulation and antibiotic biosynthesis is, therefore, a characteristic that depends on the growth physiology of the culture. Among the factors that can influence this relationship are the absolute and relative amounts of nutrients available to the organism; environmental conditions such as temperature and pH can also have metabolic consequences.