Substrate and metal complexes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae provide new insights into the catalytic mechanism

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent enzymes that catalyse the first committed step in the biosynthesis of aromatic amino acids in microorganisms and plants, the condensation of 2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP synthases are possible targets for fungicides and represent a model system for feedback regulation in metabolic pathways. To gain further insight into the role of the metal ion and the catalytic mechanism in general, the crystal structures of several complexes between the tyrosine-regulated form of DAHP synthase from Saccharomyces cerevisiae and different metal ions and ligands have been determined. The crystal structures provide evidence that the simultaneous presence of a metal ion and PEP result in an ordering of the protein into a conformation that is prepared for binding the second substrate E4P. The site and binding mode of E4P was derived from the 1.5A resolution crystal structure of DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde 3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl group of E4P replaces a water molecule coordinated to the metal ion, strongly favouring a reaction mechanism where the initial step is a nucleophilic attack of the double bond of PEP on the metal-activated carbonyl group of E4P. Mutagenesis experiments substituting specific amino acids coordinating PEP, the divalent metal ion or the second substrate E4P, result in stable but inactive Aro4p-derivatives and show the importance of these residues for the catalytic mechanism.     

The use of (E)- and (Z)-phosphoenol-3-fluoropyruvate as mechanistic probes reveals significant differences between the active sites of KDO8P and DAHP synthases

The enzymes 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-2-heptulosonate-7-phosphate (DAHP) synthase catalyze a similar aldol-type condensation between phosphoenolpyruvate (PEP) and the corresponding aldose: arabinose 5-phosphate (A5P) and erythrose 4-phosphate (E4P), respectively. While KDO8P synthase is metal-dependent in one class of organisms and metal-independent in another, only a metal-dependent class of DAHP synthases has thus far been identified in nature. We have used catalytically active E and Z isomers of phosphoenol-3-fluoropyruvate [(E)- and (Z)-FPEP, respectively] as mechanistic probes to characterize the differences and/or the similarities between the metal-dependent and metal-independent KDO8P synthases as well as between the metal-dependent KDO8P synthase and DAHP synthase. The direct evidence of the overall stereochemistry of the metal-dependent Aquifex pyrophilus KDO8P synthase (ApKDO8PS) reaction was obtained by using (E)- and (Z)-FPEPs as alternative substrates and by subsequent (19)F NMR analysis of the products. The results reveal the si face addition of the PEP to the re face of the carbonyl of A5P, and establish that the stereochemistry of ApKDO8PS is identical to that of the metal-independent Escherichia coli KDO8P synthase enzyme (EcKDO8PS). In addition, both ApKDO8PS and EcKDO8PS enzymes exhibit high selectivity for (E)-FPEP versus (Z)-FPEP, the relative k(cat)/K(m) ratios being 100 and 33, respectively. In contrast, DAHP synthase does not discriminate between (E)- and (Z)-FPEP (the k(cat)/K(m) being approximately 7 x 10(-)(3) microM(-)(1) s(-)(1) for both compounds). The pre-steady-state burst experiments for EcKDO8PS showed that product release is rate-limiting for the reactions performed with either PEP, (E)-FPEP, or (Z)-FPEP, although the rate constants, for both product formation and product release, were lower for the fluorinated analogues than for PEP [125 and 2.3 s(-)(1) for PEP, 2.5 and 0.2 s(-)(1) for (E)-FPEP, and 9 and 0.1 s(-)(1) for (Z)-FPEP, respectively]. The observed data indicate substantial differences in the PEP subsites and open the opportunity for the design of selective inhibitors against these two families of enzymes.     

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent K _m values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 M, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent K _i values of 3, 160 and 180 M, respectively. In addition, tryptophan and E4P (apparent K _i values of 11 and 530 M, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - E4P erythrose-4-phosphate - PEP phosphoenolpyruvate - RuMP ribulose monophosphate - HPLC high performance liquid chromatography - FPLC fast protein liquid chromatography - SDS sodium dodecyl sulphate     

Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities

The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non-PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.     

Substrate Ambiguity of 3-Deoxy-d-manno-Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae in the Context of Its Membership in a Protein Family Containing a Subset of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases

3-Deoxy-d-manno-octulosonate 8-phosphate (KDOP) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing reactions. The genome of Neisseria gonorrhoeae contains one gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the two nonhomologous DAHP synthase families known, the N. gonorrhoeae protein belongs to the family I assemblage. KDOP synthase exhibited an ability to replace arabinose-5-P with either erythrose-4-P or ribose-5-P as alternative substrates. The results of periodate oxidation studies suggested that the product formed by KDOP synthase with erythrose-4-P as the substrate was 3-deoxy-d-ribo-heptulosonate 7-P, an isomer of DAHP. As expected, this product was not utilized as a substrate by dehydroquinate synthase. The significance of the ability of KDOP synthase to substitute erythrose-4-P for arabinose-5-P is (i) recognition of the possibility that the KDOP synthase might otherwise be mistaken for a species of DAHP synthase and (ii) the possibility that the broad-specificity type of KDOP synthase might be a relatively vulnerable target for antimicrobial agents which mimic the normal substrates. An analysis of sequences in the database indicates that the family I group of DAHP synthase has a previously unrecognized membership which includes the KDOP synthases. The KDOP synthases fall into a subfamily grouping which includes a small group of DAHP synthases. Thus, family I DAHP synthases separate into two subfamilies, one of which includes the KDOP synthases. The two subfamilies appear to have diverged prior to the acquisition of allosteric-control mechanisms for DAHP synthases. These allosteric control specificities are highly diverse and correlate with the presence of N-terminal extensions which lack homology with one another.3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) and 3-deoxy-d-manno-octulosonate 8-phosphate (KDOP) are analogous seven- and eight-carbon 2-keto-3-deoxy sugars that are synthesized by enzymes which belong to functionally unrelated pathways. DAHP synthase forms DAHP as the acyclic precursor of the aromatic amino acids in bacteria, lower eukaryotes, and plants (3); KDOP synthase is best known for its role in the formation of KDOP as a critical component of the lipopolysaccharide of gram-negative bacteria (37), but its distribution in nature has recently been recognized to be broader (13). Both enzymes catalyze an overall condensation of phosphoenolpyruvate (PEP) with an aldose, i.e., erythrose-4-phosphate (E4P) in the case of DAHP synthase and arabinose-5-phosphate (A5P) in the case of KDOP synthase. The reactions are irreversible and are not aldol-type condensations, which unfortunately has been implied by the Enzyme Commission naming that has been recommended for DAHP synthase.As might be expected from the close structural relationship of A5P and E4P, the reactions are strikingly similar. This similarity is reflected at the level of mechanistic detail (see reference 16 and references therein). DAHP synthase and KDOP synthase, along with enolpyruvoylshikimate 3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvoyl transferase, comprise a small class of PEP-utilizing enzymes that catalyze C—O bond cleavage with respect to the release of P_i from PEP (1, 27). This contrasts with the more familiar nucleophilic attack at the phosphorous atom of PEP that results in P—O bond cleavage by the action of enzymes such as pyruvate kinase (25), PEP carboxylase (34), and PEP carboxykinase (8).In classical studies with Escherichia coli, DAHP synthase (44, 45) and KDOP synthase (41) are specific for E4P and A5P, respectively. In contrast, we found that the KDOP synthase of Neisseria gonorrhoeae possessed the ability to utilize E4P in place of A5P. We addressed the question of whether KDOP synthase of N. gonorrhoeae in the presence of E4P and PEP was able to form DAHP, in which case it would also have the potential to function as a DAHP synthase. The time-dependent cleavage of the product was investigated by the periodate-oxidation-thiobarbituric acid (TBA) assay, and these results allow some speculation on the stereospecific course of the reaction in comparison with the reaction of DAHP synthase.     

Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield.

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Purification and properties of DAHP synthase from Nocardia mediterranei

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway was isolated from Nocardia mediterranei. It has a molecular weight of approx. 135,000, and four identical subunits, each with a molecular weight of 35,000. The Km values for phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E-4-P) were 0.4 and 0.25 mM, respectively, and kinetic study showed that LTrp inhibited DAHP synthase activity, but was not competitive with respect to PEP or E-4-P. The enzyme activity was inhibited by excess of E-4-P added in the incubation system. D-ribose 5-phosphate (R-5-P), D-glucose 6-phosphate (G-6-P) or D-sedoheptulose 7-phosphate (Su-7-P) etc. inhibited DAHP synthase in cell-free extract, but on partially purified enzyme no inhibitory effect was detected. The indirect inhibition of R-5-P and other sugar phosphates was considered to be due to the formation of E-4-P catalyzed by the related enzymes present in cell-free extract.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.     

Metabolic engineering and control analysis for production of aromatics: Role of transaldolase

Aromatic metabolites in Escherichia coli and other microorganisms are derived from two common precursors: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). During growth on glucose, the levels of both E4P and PEP are insufficient for high throughput of aromatics because of the low carbon flux through the pentose pathway and the use of PEP in the phosphotransferase system. It has been shown that transketolase and PEP synthase are effective in relieving this limitation and promoting high throughput of aromatics. To determine whether transaldolase, another E4P-producing enzyme, is also a limiting factor in directing carbon flux to the aromatic pathway, E. coli transaldolase gene (tal) was cloned and overexpressed in an aroB strain which excretes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the first intermediate in the aromatic pathway. We found that overexpression of transaldolase did significantly increase the production of DAHP from glucose. This result further supports the contention that the supply of E4P is limiting when glucose is the carbon source. However, overexpression of transaldolase in strains which already overexpress transketolase did not show a further increase in production of aromatics. This result was attributed to the saturation of E4P supply when TktA was overexpressed. The flux control of DAHP production was discussed on the basis of Metabolic Control Analysis. (c) 1997 John Wiley & Sons, Inc.     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA),该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10．8倍,tktA活性提高了3．9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大(2．1～9．1倍),TktA的活性相对稳定(3．9～4．5倍),且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.     

Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine-inhibitable) of Saccharomyces cerevisiae

The phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonate-7-phosphate (dHp1P) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250-fold enrichment of the enzyme activity present in wild-type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 microM for phosphoenolpyruvate (P-pyruvate) and 130 microM for erythrose 4-phosphate (Ery4P) and the rate constant was calculated as 10 s-1. Inhibition by phenylalanine is competitive with respect to erythrose 4-phosphate and non-competitive to phosphoenolpyruvate, with a Ki of 10 microM.     

The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a gram-positive pathogen Streptococcus pneumoniae

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.     

Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations

The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations. The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius. Ammonium salts, at physiological concentrations, were dramatically more effective than other cations. Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve. Hill plots did not show detectable cooperativity in the binding of ammonium. Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P). A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated. Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation. Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P. The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP. Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.     

Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli

Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism.     

The Kinetic Mechanism of 5-Enolpyruvylshikimate-3-Phosphate Synthase from a Gram-Positive Pathogen Streptococcus Pneumoniae

Abstract

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpqruvyl group from phospho(enol)pqruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may hate potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction. GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P. suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction. GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might he formed between the enzyme. EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.     

Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions.     

Crystal structure of the reaction complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPS(Tm)) were grown in the presence of PEP and metal cofactor, Cd(2+), and then soaked with E4P at 4 degrees C where the catalytic activity of the enzyme is negligible. The crystal structure of the "frozen" reaction complex was determined at 2.2A resolution. The subunit of the DAHPS(Tm) homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (beta/alpha)(8)-barrel domain. The active site located at the C-end of the barrel contains Cd(2+), PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPS(Tm) is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPS(Ec)) and Saccharomyces cerevisiae (DAHPS(Sc)). However, the secondary, tertiary, and quaternary structures of DAHPS(Tm) are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPS(Ec) and DAHPS(Sc). Although DAHPS(Tm) is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPS(Ec) and DAHPS(Sc). A sequence similarity search revealed that DAHPSs of phylogenetic family Ibeta possess a FL domain like DAHPS(Tm) while those of family Ialpha have extra barrel segments similar to those of DAHPS(Ec) and DAHPS(Sc). This indicates that the mechanism of feedback regulation in DAHPS(Tm) and other family Ibeta enzymes is different from that of family Ialpha enzymes, most likely being mediated by the FL domain.