Enzyme 5-enolpyruvylshikimate-3-phosphate Research Articles

Integrated computer-aided drug design and biophysical simulation approaches to determine natural anti-bacterial compounds for Acinetobacter baumannii

Acinetobacter baumannii is a nosocomial bacterial pathogen and is responsible for a wide range of diseases including pneumonia, necrotizing fasciitis, meningitis, and sepsis. The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (encoded by aroA gene) in ESKAPE pathogens catalyzes the sixth step of shikimate pathway. The shikimate pathway is an attractive drug targets pathway as it is present in bacteria but absent in humans. As EPSP is essential for the A. baumannii growth and needed during the infection process, therefore it was used as a drug target herein for high-throughput screening of a comprehensive marine natural products database (CMNPD). The objective was to identify natural molecules that fit best at the substrate binding pocket of the enzyme and interact with functionally critical residues. Comparative assessment of the docking scores allowed selection of three compounds namely CMNPD31561, CMNPD28986, and CMNPD28985 as best binding molecules. The molecules established a balanced network of hydrophobic and hydrophilic interactions, and the binding pose remained in equilibrium throughout the length of molecular simulation time. Radial distribution function (RDF) analysis projected key residues from enzyme active pocket which actively engaged the inhibitors. Further validation is performed through binding free energies estimation that affirms very low delta energy of <−22 kcal/mol in MM-GBSA method and <−12 kcal/mol in MM-PBSA method. Lastly, the most important active site residues were mutated and their ligand binding potential was re-investigated. The molecules also possess good druglike properties and better pharmacokinetics. Together, these findings suggest the potential biological potency of the leads and thus can be used by experimentalists in vivo and in vitro studies.

A Review on Impact of Glyphosate on Development of Cancer

Pesticides are a vast mixture of compounds used to control pests like plants, moulds, and insects. In agriculture, non-agricultural vegetation management, and crop desiccant harvesting aid, chemicals from every major functional family of pesticides, such as insecticides, herbicides, fungicides, and fumigants, were frequently used. Herbicides are one of the most effective tools for farmers to obtain optimal crop yields when used correctly. Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum weed killer that is used all over the world in agriculture and forestry. Glyphosate's herbicidal activity in plants is to disrupt the shikimic acid pathway's generation of branched-chain amino acids by preventing the binding of phosphoenolpyruvate (PEP) to the enzyme 5-enolpyruvylshikimate 3-phosphate synthase. This causes a deficiency in aromatic amino acid synthesis and, as a result, weeds mortality. Glyphosate exposure through food, drinking water, wind, water erosion, and other environmental pathways has been linked to human health issues as a carcinogen, mutagen, and reproductive toxicity. Glyphosate has a wide range of tumorigenic effects in biological systems, and epidemiological evidence suggests that glyphosate use on crops is linked to a wide range of cancers, including liver cancer, breast cancer, thyroid cancer, pancreatic cancer, kidney cancer, bladder cancer, and myeloid cancer. The shikimate pathway enzymes, intermediates, and derivative amino acids, which have been associated to genotoxicity and carcinogenicity, are thought to have a role in most cancer pathologies. This review summarises glyphosate's function in cancer pathology, including the ability of the glyphosate circuit to induce cancer and implications for future therapeutic methods.

Novel aroA of Glyphosate-Tolerant Bacterium Pseudomonas sp. Strain HA-09 Isolated from Roundup-Contaminated Garden Soils in Iran.

Background: Glyphosate is a non-selective systemic herbicide with a broad spectrum of weed control that inhibits a key enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in the shikimate pathway.Objectives: Isolation and analysis of the epsps (aroA) gene responsible for glyphosate-tolerance in bacteria from Roundup- contaminated soils was the aim of this study.Materials and Methods: Sampling was done from the soil of the gardens which were heavily contaminated by Roundup herbicide and then bacterial screening was performed in the presence of high concentrations of glyphosate. The genus of bacterium was identified via molecular methods such as 16S rRNA sequencing. The aroA gene of this bacterium (aroAHA-09) was isolated using the primers designed-upon specific regions of aroA genes available in NCBI GenBank database. The PCR product was cloned, sequenced and subcloned into pET28a as an expression vector and transferred into E. coli strain BL21(DE3). The cells were inoculated in liquid M9 minimal medium containing IPTG and different concentrations of glyphosate.Results: The genus of bacterium was identified as Pseudomonas sp. strain HA-09. The isolated aroAHA-09 gene from this bacterium was approximately 2.2 kb in size. Bioassay of E. coli expressing this gene showed high tolerance to glyphosate (up to 300 mM).Conclusion: The aroAHA-09 gene could be considered as a novel and efficient candidate for development of glyphosate-tolerant crop plants.

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

Divergent properties and phylogeny of cyanobacterial 5-enol-pyruvyl-shikimate-3-phosphate synthases: evidence for horizontal gene transfer in the Nostocales.

As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples.

A red and far‐red light receptor mutation confers resistance to the herbicide glyphosate

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

Screening of Photosynthetic Pigments for Herbicidal Activity with a New Computational Molecular Approach

There is an immense interest among the researchers to identify new herbicides which are effective against the herbs without affecting the environment. In this work, photosynthetic pigments are used as the ligands to predict their herbicidal activity. The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a good target for the herbicides. Homology modeling of the target enzyme is done using Modeler 9.11 and the model is validated. Docking studies were performed with AutoDock Vina algorithm to predict the binding of the natural pigments such as β-carotene, chlorophyll a, chlorophyll b, phycoerythrin and phycocyanin to the target. β-carotene, phycoerythrin and phycocyanin have higher binding energies indicating the herbicidal activity of the pigments. This work reports a procedure to screen herbicides with computational molecular approach. These pigments will serve as potential bioherbicides in the future.

A novel RPMXR motif among class II 5-enolpyruvylshikimate-3-phosphate synthases is required for enzymatic activity and glyphosate resistance

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is an attractive target for drugs and herbicides. Here we identified a novel RPMXR motif that is strictly conserved among class II EPSP synthases. Site-directed mutational analysis of this motif showed that substitutions of the four strictly conserved amino acid residues, Arg127, Pro128, Met129, and Arg131, resulted in complete loss of enzymatic activity, whereas changes in the non-conserved Asn130 residue strongly influenced glyphosate resistance (all numbering according to Pseudomonas stutzeri A1501 EPSP synthase). These experimental results, combined with 3D structure modeling of the location and interaction of the RPMXR motif with phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P), demonstrate that the novel motif is required for enzymatic activity and glyphosate resistance of class II EPSP synthases.

A simple chromatographic assay to discriminate between glyphosate-resistant and susceptible soybean ( Glycine max) cultivars

In glyphosate-susceptible soybean ( Glycine max L. Merrill), the herbicide glyphosate [( N-phosphonomethyl)glycine] inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, causing a massive accumulation of the metabolite shikimate. This phenomenon does not occur in glyphosate-resistant soybean due the presence of a gene encoding glyphosate-insensitive EPSP synthase. This study proposes a simple and reliable assay as an alternative tool for differentiating glyphosate-resistant from susceptible soybean cultivars. The assay is based on a single extraction of leaf or root tissue. Shikimate is quantified by reversed-phase high–performance liquid chromatography at 220 nm and isocratic elution with phosphoric acid. After glyphosate treatment, tissues of glyphosate-susceptible plants were found to massively accumulate shikimate, whereas the tissues of glyphosate-resistant plants did not accumulate the metabolite.

Molecular basis for the herbicide resistance of Roundup Ready crops.

The engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100-Gly), allowing glyphosate to bind in its extended, inhibitory conformation.

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.

How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli.

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.

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 x10(4) clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

Isolation and biochemical diagnosis of cell lines of groundnut (Arachis hypogaeaL) selected on glyphosate†

Three cell lines of groundnut (Arachis hypogaea L), an important oilseed legume, were selected on glyphosate using in-vitro culture techniques. The cell lines isolated through single as well as stepwise selection procedures showed c 20-fold increase in glyphosate tolerance as compared to the unselected control cell line. Studies on the biochemical mechanism of glyphosate tolerance in these cell lines showed a significant increase in the total extractable activity of the target enzyme, 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), which was further confirmed with immunological data. The over-expressed EPSP synthase activity was, however, subject to inhibition by glyphosate in vitro. Two other key regulated enzymes of the shikimic acid pathway, 3-deoxy-D-arabino heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) and chorismate mutase (CM) (EC 5.4.99.5) did not show any change in specific activity in the selected cell lines. The enhanced activity of EPSP synthase in the tolerant cell lines was found to be stably inherited in the absence of selection pressure. © 1999 Society of Chemical Industry

Evidence for the shikimate pathway in apicomplexan parasites.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

Structural Constraints on the Ternary Complex of 5-Enolpyruvylshikimate-3-phosphate Synthase from Rotational-echo Double-resonance NMR

The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phospho enolpyruvate to form EPSP. The reaction is inhibited by N-(phosphono- methyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP synthase and distances to a 13C label in Glp and to the 31P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.

Ligand geometry of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) 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. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development.

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W10BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.

5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties).

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.