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1.
Isothermal titration calorimetry measurements are reported which give important new binding constant (Kd) information for various substrate and inhibitor complexes of Escherichia coli EPSP synthase (EPSPS). The validity of this technique was first verified by determining Kd's for the known binary complex with the substrate, shikimate 3-phosphate (S3P), as well as the herbicidal ternary complex with S3P and glyphosate (EPSPS.S3P.glyphosate). The observed Kd's agreed very well with those from previous independently determined kinetic and fluorescence binding measurements. Further applications unequivocally demonstrate for the first time a fairly tight interaction between phosphoenolpyruvate (PEP) and free enzyme (Kd = 390 microM) as well as a correspondingly weak affinity for glyphosate (Kd = 12 mM) alone with enzyme. The formation of the EPSPS.PEP binary complex was independently corroborated using equilibrium dialysis. These results strongly suggest that S3P synergizes glyphosate binding much more effectively than it does PEP binding. These observations add important new evidence to support the hypothesis that glyphosate acts as a transition-state analogue of PEP. However, the formation of a catalytically productive PEP binary complex is inconsistent with the previously reported compulsory binding order process required for catalysis and has led to new studies which completely revise the overall EPSPS kinetic mechanism. A previously postulated ternary complex between S3P and inorganic phosphate (EPSPS.S3P.Pi, Kd = 4 mM) was also detected for the first time. Quantitative binding enthalpies and entropies were also determined for each ligand complex from the microcalorimetry data. These values demonstrate a clear difference in thermodynamic parameters for recognition at the S3P site versus those observed for the PEP, Pi, and glyphosate sites. 相似文献
2.
Priestman MA Healy ML Becker A Alberg DG Bartlett PA Lushington GH Schönbrunn E 《Biochemistry》2005,44(9):3241-3248
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. 相似文献
3.
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. 相似文献
4.
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. 相似文献
5.
The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements. Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone. By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM). The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration. At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM. Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G. represents the complex with altered fluorescence. The kinetics of binding were measured by stopped-flow fluorescence methods. The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶的酶学性质 总被引:1,自引:0,他引:1
核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSP合酶)是AROM多功能酶的活性之一.该酶催化莽草酸磷酸(S3P)和磷酸烯醇式丙酮酸(PEP)产生5-烯醇丙酮酰莽草酸-3-磷酸和无机磷酸的可逆反应,受除草剂草甘膦(N-(膦羧甲基)甘氨酸)抑制.纯化了核盘菌AROM蛋白并对EPSP合酶进行了酶学特征研究.结果显示,该酶反应的最适pH值为7.2,最适温度为30℃.热失活反应活化能是69.62 kJ/mol.底物S3P和PEP浓度分别高于1 mmol/L和2 mmol/L时,对EPSP合酶反应产生抑制作用.用双底物反应恒态动力学Dalziel方程求得的Km(PEP)为140.98 μmol/L,K m(S3P)为139.58 μmol/L.酶动力学模型遵循顺序反应机制.草甘膦是该酶反应底物PEP的竞争性抑制剂(Ki为0.32 μmol/L)和S3P的非竞争性抑制剂.正向反应受K+激活.当[K+]增加时,K m(PEP)随之降低,Km(S3P)不规律变化,而K i(PEP)随[K+]增加而提高. 相似文献
7.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):571-581
AbstractThe 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. 相似文献
8.
S R Padgette C E Smith Q K Huynh G M Kishore 《Archives of biochemistry and biophysics》1988,266(1):254-262
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. 相似文献
9.
5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate 总被引:2,自引:0,他引:2
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. 相似文献
10.
A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics 总被引:1,自引:0,他引:1
Direct evidence for an enzyme-bound intermediate in the EPSP synthase reaction pathway has been obtained by rapid chemical quench-flow studies. The transient-state kinetic analysis has led to the following complete scheme: (formula; see text) Values for all 12 rate constants were obtained. Substrate trapping experiments in the forward and reverse reactions established the kinetically preferred order of binding and release of substrates and products and showed that shikimate 3-phosphate (S3P) and 5-enolpyruvoylshikimate 3-phosphate (EPSP) dissociate at rates greater than turnover in each direction. Pre-steady-state bursts of product formation were observed in the reaction in each direction indicating a rate-limiting step following catalysis. Single turnover experiments with enzyme in excess over substrate demonstrated the formation of a transient intermediate in both the forward and reverse reactions. In these experiments, the enzymatic reaction was observed by employing a radiolabel in the enol moiety of either phosphoenol pyruvate (PEP) or EPSP. The separation and quantitation of reaction products were accomplished by HPLC monitoring radioactivity. The intermediate was observed as the transient production of radiolabeled pyruvate, formed due to the breakdown of the intermediate in the acid quench used to stop the reaction. The intermediate was observed within 5-10 ms after the substrates were mixed with enzyme and decayed in a reaction paralleling the formation of product in each direction. Thus, the kinetics demonstrate directly the kinetic competence of the presumed intermediate. No pyruvate was formed, on a time scale which is relevant to catalysis, after incubation of the enzyme with dideoxy-S3P and PEP or with EPSP in the absence of phosphate; and so, the intermediate does not accumulate under these conditions. The intermediate broke down to form PEP and EPSP in addition to pyruvate when the reaction was quenched with base rather than acid; therefore, the intermediate must contain the elements of each product. Other experiments were designed to measure directly the phosphate binding rate and further constrain the PEP binding rate. The overall solution equilibrium constant in the forward direction was determined to be 180 by quantitation of radiolabeled reactants and products in equilibrium after incubation with a low enzyme concentration. The internal, active site equilibrium constant was obtained by incubation of radiolabeled S3P with excess enzyme and high concentrations of phosphate and PEP to provide the ratio of [EPSP]/[S3P] = 2.3, which is largely a measure of K4.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy 总被引:9,自引:0,他引:9
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5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops. 相似文献
12.
Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site 总被引:11,自引:0,他引:11
S R Padgette D B Re C S Gasser D A Eichholtz R B Frazier C M Hironaka E B Levine D M Shah R T Fraley G M Kishore 《The Journal of biological chemistry》1991,266(33):22364-22369
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. 相似文献
13.
5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae. 1. Purification and properties 总被引:5,自引:0,他引:5
The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62-1 (= Klebsiella pneumoniae ATCC 25306). A 3300-fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE-cellulose, Sephadex G-75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%. An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr = 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme-catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3-phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60 degrees C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations. Non-linear Lineweaver-Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme-substrate complexes: forward reaction: 43 microM (PEP) and 22 microM (S3P); reverse reaction: 1.3 microM (EPSP) and 2.6 mM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction. 相似文献
14.
Marques MR Vaso A Neto JR Fossey MA Oliveira JS Basso LA dos Santos DS de Azevedo Junior WF Palma MS 《Biochemistry》2008,47(28):7509-7522
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. 相似文献
15.
Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase 总被引:1,自引:0,他引:1
Healy-Fried ML Funke T Priestman MA Han H Schönbrunn E 《The Journal of biological chemistry》2007,282(45):32949-32955
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. 相似文献
16.
How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli 总被引:2,自引:0,他引:2
The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site. 相似文献
17.
R S Fischer J L Rubin C G Gaines R A Jensen 《Archives of biochemistry and biophysics》1987,256(1):325-334
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. 相似文献
18.
UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme. 相似文献
19.
Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate 总被引:1,自引:0,他引:1
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. 相似文献
20.
Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells 总被引:6,自引:0,他引:6
Yu-Yau Joanne Shyr Angus G. Hepburn Jack M. Widhohn 《Molecular & general genetics : MGG》1992,232(3):377-382
Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- DTT
dithiothreitol
- EPSPS
5-enolpyruvylshikimate-3-phosphate synthase
- I50
50% inhibitory concentration
- Kb
Kilobase (pairs)
- PEP
phosphoenolpyruvate
- PMSF
phenylmethylsulfonyl fluoride
- PVPP
polyvinylpolypyrrolidone
- S-3-P
shikimate-3-phosphate 相似文献