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.     

Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 mM glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogeneous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with Mr 49,000-55,800. Km values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 18 microM, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate (Ki = 0.17 microM). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.     

Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase by o-phthalaldehyde

In order to identify the essential reactive amino acid residues of 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosphate (N-phosphonomethylglycine), chemical modification studies with o-phthalaldehyde were undertaken. Incubation of the enzyme with the reagent resulted in a time-dependent loss of enzyme activity. The inactivation followed first-order and saturation kinetics with a Kinact of 25 microM and a maximum rate constant of 0.34 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvoylshikimate 3-phosphate, or by a combination of shikimate 3-phosphate plus glyphosate, but not by phosphoenolpyruvate or glyphosate alone. Absorbance and fluorescence spectra studies indicate that complete inactivation of the enzyme resulted from the formation of two isoindole derivatives per molecule of enzyme. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate resulted in the isolation of two peptides which were not found for the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. Analyses of these two peptides indicate that Lys-22 and Lys-340 were the modified sites. The amino acid sequences around these residues are conserved in bacterial, fungal, as well as plant enzymes, suggesting that these regions may constitute part of the enzyme active site.     

核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶的酶学性质

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.     

5-Enolpyruvyl shikimate 3-phosphate synthase from Escherichia coli. Identification of Lys-22 as a potential active site residue

5-Enolpyruvyl shikimate 3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enolpyruvyl shikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). In order to determine the role of lysine residues in the mechanism of action of this enzyme as well as in its inhibition by glyphosate, chemical modification studies with pyridoxal 5'-phosphate were undertaken. Incubation of the enzyme with the reagent in the absence of light resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order and saturation kinetics with Kinact of 45 microM and a maximum rate constant of 1.1 min-1. The inactivation rate increased with increase in pH, with a titratable pK of 7.6. Activity of the inactive enzyme was restored by addition of amino thiol compounds. Reaction of enzyme with pyridoxal 5'-phosphate was prevented in the presence of substrates or substrate plus glyphosate, an inhibitor of the enzyme. Upon 90% inactivation, approximately 1 mol of pyridoxal 5'-phosphate was incorporated per mol of enzyme. The azomethine linkage between pyridoxal 5'-phosphate and the enzyme was reduced by NaB3H4. Tryptic digestion followed by reverse phase chromatographic separation resulted in the isolation of a peptide which contained the pyridoxal 5'-phosphate moiety as well as 3H label. By amino acid sequencing of this peptide, the modified residue was identified as Lys-22. The amino acid sequence around Lys-22 is conserved in bacterial, fungal, as well as plant enzymes suggesting that this region may constitute a part of the enzyme's active site.     

A radiometric assay for 5-enolpyruvylshikimate-3-phosphate synthase

A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [¹⁴C]shikimate 3-phosphate from the commercially available precursor [¹⁴C]shikimic acid is also described.     

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.     

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.     

Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate

The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K(i) 1.1 microM, and uncompetitive with respect to shikimate-3-phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate-3-phosphate complex.     

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.     

Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate

Abstract The potent inhibition of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase by the broad-spectrum herbicide glyphosate ( N -[phosphonomethyl]glycine) was confirmed for the enzymes extracted from various bacteria, a green alga and higher plants. However, 5 out of 6 species belonging to the genus Pseudomonas were found to have EPSP synthases with a 50- to 100-fold decreased sensitivity to the inhibitor. Correspondingly, growth of these 5 species was not inhibited by 5 mM glyphosate, and the organisms did not excrete shikimate-3-phosphate in the presence of the herbicide.     

N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase.

Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40-fold accumulation of the target enzyme. 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system. In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethyl)glycine.     

Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.     

5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

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.     

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.     

A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.     

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.     

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

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     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.