Interaction of phosphonate analogues of the tetrahedral reaction intermediate with 5-enolpyruvylshikimate-3-phosphate synthase in atomic detail

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway and is the target of the broad-spectrum herbicide glyphosate. Since the functionality of the shikimate pathway is vital not only for plants but also for microorganisms, EPSPS is considered a prospective target for the development of novel antibiotics. We have kinetically analyzed and determined the crystal structures of Escherichia coli EPSPS inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate. Both diastereomers are competitive inhibitors with respect to the substrates of the EPSPS reaction, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). Remarkably, the (S)-phosphonate (K(iS3P) = 750 nM), whose configuration corresponds to that of the genuine tetrahedral intermediate, is a much weaker inhibitor than the (R)-phosphonate analogue (K(iS3P) = 16 nM). The crystal structures of EPSPS liganded with the (S)- and (R)-phosphonates, at 1.5 and 1.9 A resolution, respectively, revealed that binding of the (R)-phosphonate induces conformational changes of the strictly conserved residues Arg124 and Glu341 within the active site. This appears to give rise to substantial structural alterations in the amino-terminal globular domain of the enzyme. By contrast, binding of the (S)-phosphonate renders the enzyme structure unchanged. Thus, EPSPS may facilitate the tight binding of structurally diverse ligands through conformational flexibility. Molecular docking calculations did not explain why the (R)-phosphonate is the better inhibitor. Therefore, we propose that the structural events during the open-closed transition of EPSPS are altered as a result of inhibitor action.     

Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.     

Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 Angstroms resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP synthase enzymes are discussed.     

Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state

The shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor-liganded and tetrahedral intermediate (TI)-liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI-liganded structure provides absolute configuration of the C-2 atom from the F-PEP moiety of the enzyme-bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate-bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new-generation antibiotics.     

5-Enolpyruvylshikimate-3-phosphate synthase: determination of the protonation state of active site residues by the semiempirical method

EPSP synthase (EPSPS) catalyzes the addition of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (TI) that is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. A semiempirical molecular modeling study of the EPSPS active site containing the TI was implemented for the assignment of the protonation states of four basic residues, Lys22, Lys340, His385, and Lys411, based on the evaluation of 16 different protonation states and comparison of the resulting energy minimized heavy atoms coordinates with available X-ray crystallographic data of the D313A mutant of EPSPS. The results, employing both gas phase and continuum solvent models, are indicative that after the TI formation the histidine residue is most probably in neutral form (N^ε-protonated) and the lysine residues are in protonated form, which suggests that none of the presently proposed assignments of aminoacid residues involved in the reaction mechanism could be completely correct. The protonated state of Lys22 in the presence of the TI supports the proposal that this residue is a general acid catalyst for TI breakdown. Modeling of the native enzyme active site suggests that Asp313 residue has only minor effects on the definition of the TI position inside the active site. Hydrogen-bonds distances suggest that, in order to act as a base, Asp313 needs the intermediacy of a hydroxyl group of the TI for effecting the attack on the TI methyl group in the elimination step leading to EPSP, as suggested previously in the literature.     

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.     

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis ( Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.     

Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli.

Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [Ki(PEP) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat. Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.     

Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R.aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E.coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R.aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E.coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.     

Structural analysis of chorismate synthase from Plasmodium falciparum: a novel target for antimalaria drug discovery

The shikimate pathway in Plasmodium falciparum provides several targets for designing novel antiparasitic agents for the treatment of malaria. Chorismate synthase (CS) is a key enzyme in the shikimate pathway which catalyzes the seventh and final step of the pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS. The structure of PfCS along with cofactor FMN was predicted by homology modeling using crystal structure of Helicobacter pylori chorismate synthase (HpCS). The quality of the model was validated using structure analysis servers and molecular dynamics. Dimeric form of PfCS was generated and the FMN binding mechanism involving movement of loop near active site has been proposed. Active site pocket has been identified and substrate 5-enolpyruvylshikimate 3-phosphate (EPSP) along with screened potent inhibitors has been docked. The study resulted in identification of putative inhibitors of PfCS with binding efficiency in nanomolar range. The selected putative inhibitors could lead to the development of anti-malarial drugs.     

Overproduction of,and interaction within,bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors

The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.     

Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase

Glyphosate, the world's most used herbicide, is a massive success because it enables efficient weed control with minimal animal and environmental toxicity. The molecular target of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the sixth step of the shikimate pathway in plants and microorganisms. Glyphosate-tolerant variants of EPSPS constitute the basis of genetically engineered herbicide-tolerant crops. A single-site mutation of Pro(101) in EPSPS (numbering according to the enzyme from Escherichia coli) has been implicated in glyphosate-resistant weeds, but this residue is not directly involved in glyphosate binding, and the basis for this phenomenon has remained unclear in the absence of further kinetic and structural characterization. To probe the effects of mutations at this site, E. coli EPSPS enzymes were produced with glycine, alanine, serine, or leucine substituted for Pro(101). These mutant enzymes were analyzed by steady-state kinetics, and the crystal structures of the substrate binary and substrate.glyphosate ternary complexes of P101S and P101L EPSPS were determined to between 1.5- and 1.6-A resolution. It appears that residues smaller than leucine may be substituted for Pro(101) without decreasing catalytic efficiency. Any mutation at this site results in a structural change in the glyphosate-binding site, shifting Thr(97) and Gly(96) toward the inhibitor molecule. We conclude that the decreased inhibitory potency observed for glyphosate is a result of these mutation-induced long-range structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Arginine chemical modification of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase

Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.     

Phosphate analogues as probes of the catalytic mechanisms of MurA and AroA, two carboxyvinyl transferases

The role in catalysis of phosphate with AroA (enolpyruvyl shikimate 3-phosphate synthase) and MurA (enolpyruvyl UDP-GlcNAc synthase) was probed using phosphate analogues and an AroA mutant. Phosphate, the second reaction product, increases the reactivity of the enolpyruvyl products (EP-OR's) approximately 10(5)-fold in the reverse reaction, forming phosphoenolpyruvate and R-OH (shikimate 3-phosphate or UDP-GlcNAc). Phosphate is intrinsically unreactive with EP-OR, raising the question of how AroA and MurA promote EP-OR reactivity. Eleven phosphate analogues were examined. All those with tetrahedral geometries bound with AroA, except sulfate, while no nontetrahedral analogues did. Arsenate, vanadate, and fluorophosphate caused reactions of AroA and MurA with EP-OR's, yielding pyruvate and R-OH. Their k(cat)/K(M) values relative to phosphate were similar for both enzymes, ca. 100-fold worse for arsensate, 200-fold worse for vanadate, and 5000-fold worse for fluorophosphate, implying similar interactions with both enzymes. Examination of the arsenate-promoted reactions using [3'-(3)H]EP-OR's, (2)H(2)O, and H(2)(18)O provided evidence of an arseno-tetrahedral intermediate, analogous to the natural tetrahedral intermediate, proceeding to arsenoenolpyruvate, which spontaneously broke down to pyruvate and arsenate. The only physicochemical property that appeared to be essential for reactivity of the analogues was the presence of a proton. Titration of the intrinsic tryptophan fluorescence of the weakly active AroA mutant, Asp313Ala (D313A), demonstrated a fluorescence decrease upon enolpyruvyl shikimate 3-phosphate (EPSP) binding, and a further decrease upon binding of phosphate or arsenate to AroA_D313A.EPSP, suggesting a further conformational change. We are hopeful that understanding enzyme-phosphate interactions will make it possible to design inhibitors that can use the high endogenous phosphate concentration in bacteria to enhance inhibitor binding.     

Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate

Cultured cells of the higher plant Corydalis sempervirens Pers. which had been adapted to growing in the presence of 5 mM glyphosate (N-[phosphonomethyl]-glycine), a herbicide and a potent specific inhibitor of the shikimate pathway enzyme 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, had a nearly 40-fold increased level of the extractable activity of EPSP synthase. Activities of five other shikimate pathway enzymes were, however, similar in the adapted and nonadapted cells, and the concentrations of the free aromatic amino acids in the two cell lines were also similar. EPSP synthases purified from glyphosate-adapted, as well as nonadapted cells, had identical physical, kinetic, and immunological properties, which indicated that the glyphosate-sensitive enzyme was overproduced in the adapted culture. Overproduction of EPSP synthase in the adapted culture was unequivocally established by two-dimensional polyacrylamide gel electrophoresis, as well as by one-dimensional sodium dodecyl sulfate-gradient gel electrophoresis and quantitation of EPSP protein by immunoassay after transfer to nitrocellulose membranes. While about 0.06% of the total soluble protein from nonadapted cells was EPSP synthase protein, the proportion was 2.6% in the adapted cells. In vivo pulse-labeling experiments with [35S]methionine established that the adapted cells have an increased rate of EPSP synthase protein synthesis.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate