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...

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1.

The CREATE-ECLA Randomized Controlled TrialThe CREATE-ECLA Trial Group Investigators* See also pp 427 and 489.

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