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.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

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.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.     

Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

Summary 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.     

Mode of action of glyphosate in Candida maltosa

The broad-spectrum herbicide glyphosate inhibits the growth of Candida maltosa and causes the accumulation of shikimic acid and shikimate-3-phosphate. Glyphosate is a potent inhibitor of three enzymes of aromatic amino acid biosynthesis in this yeast. In relation to tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase, the inhibitory effect appears at concentrations in the mM range, but 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase is inhibited by micromolar concentrations of glyphosate. Inhibition of partially purified EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate (PEP) with a K _i -value of 12 M. The app. K _m for PEP is about 5-fold higher and was 62 M. Furthermore, the presence of glyphosate leads to derepression of many amino acid biosynthetic enzymes.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - EPSP synthase 5-enolpyruvylshikimate 3-phosphate synthase - PEP phosphoenolpyruvate - S-3-P shikimate-3-phosphate     

Insensitivity of 5-enolpyruvylshikimic acid-3-phosphate synthase to glyphosate confers resistance to this herbicide in a strain of Aerobacter aerogenes

The broadspectrum herbicide glyphosate (N-[phosphonomethyl]glycine), an inhibitor of the shikimate pathway enzyme 5-enolpyruvyl-shikimic acid-3-phosphate (EPSP)-synthase, inhibits the growth of Aerobacter aerogenes and causes the excretion of shikimic acid-3-phosphate. A strain of A. aerogenes, resistant to inhibition of growth by glyphosate, was isolated and found to have a glyphosate-insensitive EPSP-synthase and to no longer excrete shikimic acid-3-phosphate in the presence of glyphosate. Partial identity of EPSP-synthases from the glyphosate-sensitive and-resistant A. aerogenes strains was demonstrated by immunological procedures.Abbreviation EPSP-synthase 5-enolpyruvylshikimic acid-3-phosphate synthase (EC 2.5.1.19; 3-phosphoshikimate 1-carboxyvinyltransferase)     

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     

Impairment of carbon metabolism induced by the herbicide glyphosate

The herbicide glyphosate reduces plant growth and causes plant death by inhibiting the biosynthesis of aromatic amino acids. The objective of this work was to determine whether glyphosate-treated plants show a carbon metabolism pattern comparable to that of plants treated with herbicides that inhibit branched-chain amino acid biosynthesis. Glyphosate-treated plants showed impaired carbon metabolism with an accumulation of carbohydrates in the leaves and roots. The growth inhibition detected after glyphosate treatment suggested impaired metabolism that impedes the utilization of available carbohydrates or energy at the expected rate. These effects were common to both types of amino acid biosynthesis inhibitors. Under aerobic conditions, ethanolic fermentative metabolism was enhanced in the roots of glyphosate-treated plants. This fermentative response was not related to changes in the respiratory rate or to a limitation of the energy charge. This response, which was similar for both types of herbicides, might be considered a general response to stress conditions.     

Genetic analysis of glyphosate tolerance in Halomonas variabilis strain HTG7

Summary A highly glyphosate-tolerant bacterium strain HTG₇ was isolated from glyphosate-polluted soil in north China, and identified as Halomonas variabilis. It was a Gram-negative motile rod giving convex colony. The strain HTG₇ could tolerate up to 900 mM glyphosate in minimal medium. The 16S rDNA sequence was amplified by PCR using universal primers. The region essential for glyphosate tolerance was localized to a 3.5-kb fragment from a cosmid library of HTG₇. The DNA fragment consisted of one complete open reading frame (ORF) and one partial ORF. The partial ORF was homologous to prephenate dehydrogenase of Pseudomonas aeruginosa PA01. The complete ORF contained the tyrA and aroA genes. Only the 1.35-kb aroA encoding EPSP synthase conferred glyphosate tolerance, and complemented with E. coli aroA mutant ER2799. E. coli JM109 harboring aroA grew well in Mops supplemented with 80 mM glyphosate.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Characterization of a glyphosate-insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase

A glyphosate (N-[phosphonomethyl]glycine)-insensitive 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase has been purified from a strain of Klebsiella pneumoniae which is resistant to this herbicide [(1984) Arch. Microbiol. 137, 121-123] and its properties compared with those of the glyphosate-sensitive EPSP synthase of the parent strain. The apparent K_m values of the insensitive enzyme for phosphoenolpyruvate (PEP) and shikimate 3-phosphate (S-3-P) were increased 15.6- and 4.3-fold, respectively, as compared to those of the sensitive enzyme, and significant differences were found for the optimal pH and temperature, as well as the isoelectric points of the two enzymes. While PEP protected both enzymes against inactivation by N-ethylmaleimide, 3-bromopyruvate, and phenylglyoxal, glyphosate protected only the sensitive enzyme.     

The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli

The Escherichia coli aroA gene which codes for the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) has been cloned from the lambda-transducing bacteriophage lambda pserC. The gene has been located on a 4.7 kilobase pair PstI DNA fragment which has been inserted into the multiple copy plasmid pAT153. E. coli cells transformed with this recombinant plasmid overproduce EPSP synthase 100-fold. A simple method for the purification of homogeneous enzyme in milligram quantities has been devised. The resulting enzyme is indistinguishable from enzyme isolated from untransformed E. coli.     

Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) from shoot tissue of pea seedlings was purified to apparent homogeneity by sequential ammonium-sulphate precipitation, ion-exchange and hydrophobic-interaction chromatography and substrate elution from cellulose phosphate. Gel electrophoresis and gel-permeation chromatography showed that the purified enzyme was monomeric with molecular weight 50,000. The herbicide glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction.Abbreviations DEAE diethylaminoethyl - EPSP 5-enolpyruvylshikimate 3-phosphate     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.     

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.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview

The methylerythritol phosphate pathway to isoprenoids, an alternate biosynthetic route present in many bacteria, algae, plants, and the malarial parasite Plasmodium falciparum, has become an attractive target for the development of new antimalarial and antibacterial compounds. The second enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267), has been shown to be the molecular target for fosmidomycin, a promising antimalarial drug. This enzyme converts 1-deoxy-D-xylulose 5-phosphate (DXP) into the branched compound 2-C-methyl-D-erythritol 4-phosphate (MEP). The transformation of DXP into MEP requires an isomerization, followed by a NADPH-dependent reduction. The discovery of DXR, its subsequent characterization, and the identification of inhibitors will be presented.     

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.