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.conformational change ͉ crystal structure ͉ genetic modification ͉ mutation T he broad-spectrum herbicide glyphosate, the active ingredient of Roundup, inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the enzyme catalyzing the penultimate step of the shikimate pathway toward the biosynthesis of aromatic amino acids. Roundup Ready crop lines contain a gene derived from Agrobacterium sp. strain CP4, encoding a glyphosate-tolerant enzyme, the so-called CP4 EPSP synthase (1, 2). Expression of CP4 EPSP synthase results in glyphosate-tolerant crops, enabling more effective weed control by allowing postemergent herbicide application. The substantial advantages of glyphosate-tolerant crops have resulted in rapid adoption: 87% of soybeans, 61% of cotton, and 26% of corn planted in the United States in 2005 were glyphosate-tolerant varieties (3). However, lingering concerns about the potential health and environmental effects of genetically modified organisms have limited the acceptance of such seed lines and food products, particularly in Europe and Japan.EPSP synthase catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) (Fig. 1A). Beginning in the early 1980s, researchers sought to identify glyphosate-insensitive EPSP synthases that could be introduced into crops to provide herbicide resistance. A number of promising enzymes were identified through selective evolution, site-directed mutagenesis, and microbial screens (4-7). However, an increased tolerance for glyphosate in EPSP synthase was often accompanied by a concomitant decrease in the enzyme's affinity for PEP, resulting in decreased catalytic efficiency. More favorable kinetic characteristics were observed in some enzymes with substitutions including Pro-101-Ser and Thr-97-Ile (nu...

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K i ‫؍‬ 2.4 mM) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K m ‫؍‬ 0.1 mM). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly 96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile 97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr 97 and Pro 101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.Glyphosate (N-phosphonomethylglycine) is a potent inhibitor of the shikimate pathway in plants, specifically targeting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, 3 EC 2.5.1.19) (1). Glyphosate-based formulations exhibit broad spectrum herbicidal activity with minimal human and environmental toxicity (2, 3). The safety and efficacy of glyphosate, together with the existence of genetically modified, glyphosate-resistant crop varieties (4, 5), have combined to make glyphosate the most used herbicide in the world. Enzymes of the shikimate pathway are also regarded as attractive antimicrobial targets (6 -9).EPSPS catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxy position of shikimate-3-phosphate (S3P) (Fig. 1). Binding of the first substrate, S3P, to the enzyme triggers a global conformational change from an "open" to a "closed" conformation. PEP and glyphosate bind in the active site, formed at the interface between the Nand C-terminal globular domains. Glyphosate inhibition is competitive with respect to PEP (10, 11), and structural studies confirmed that glyphosate occupies the PEP-binding site (12-15).EPSPS from different organisms have been divided into two classes according to intrinsic glyphosate sensitivity: in Class I enzymes, found in all plants and i...

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 substrate⅐glyphosate ternary complexes of P101S and P101L EPSPS were determined to between 1.5-and 1.6-Å 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 longrange structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.Glyphosate (N-phosphonomethylglycine) inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS 2 ; EC 2.5.1.19) (1), which is essential for the biosynthesis of aromatic compounds in plants, fungi, bacteria, and apicomplexan parasites (2-5). Glyphosate, the active ingredient in Roundup, exhibits broad-spectrum herbicidal activity, yet is essentially nontoxic to animals and does not persist in the environment. These characteristics have made it the world's most popular herbicide, and usage continues to increase with the adoption of glyphosate-dependent technologies, including herbicide-tolerant crops and minimal tillage (no-till) agriculture. The enormous reliance on glyphosate and the absence of suitably safe alternative herbicides mean that the widespread emergence of glyphosatetolerant weeds would have devastating agricultural and environmental consequences. EPSPS, the molecular target of glyphosate, catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (P-enolpyruvate) to the 5-hydroxy position of shikimate 3-phosphate (S3P) (see Fig. 1). The structure of the glyphosate-inhibited complex shows that glyphosate binds to the P-enolpyruvate-binding site of EPSPS (6 -8), corroborating early kinetic data demonstrating that glyphosate binding is competitive with respect to P-enolpyruvate (1, 9, 10). Before bacte...

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) is best known as the target of the herbicide glyphosate. EPSPS is also considered an attractive target for the development of novel antibiotics since the pathogenicity of many microorganisms depends on the functionality of the shikimate pathway. Here, we have investigated the inhibitory potency of stable fluorinated or phosphonate-based analogues of the tetrahedral reaction intermediate (TI) in a parallel study utilizing class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSPS. The (R)-difluoromethyl and (R)-phosphonate analogues of the TI are the most potent inhibitors of EPSPS described to date. However, we found that class II EPSPS are up to 400 times less sensitive to inhibition by these TI analogues. X-ray crystallographic data revealed that the conformational changes of active site residues observed upon inhibitor binding to the representative class I EPSPS from Escherichia coli do not occur in the prototypical class II enzyme from Agrobacterium sp. strain CP4. It appears that because the active sites of class II EPSPS do not possess the flexibility to accommodate these TI analogues, the analogues themselves undergo conformational changes, resulting in less favorable inhibitory properties. Since pathogenic microorganisms such as Staphylococcus aureus utilize class II EPSPS, we conclude that the rational design of novel EPSPS inhibitors with potential as broad-spectrum antibiotics should be based on the active site structures of class II EPSP synthases.