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J. Biol. Chem., Vol. 282, Issue 45, 32949-32955, November 9, 2007

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Structural Basis of Glyphosate Tolerance Resulting from Mutations of Pro¹⁰¹ in Escherichia coli 5-Enolpyruvylshikimate-3-phosphate Synthase^*

From the Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045

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 Pro¹⁰¹ 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 Pro¹⁰¹. These mutant enzymes were analyzed by steady-state kinetics, and the crystal structures of the substrate binary and substrate·glyphosate 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 Pro¹⁰¹ without decreasing catalytic efficiency. Any mutation at this site results in a structural change in the glyphosate-binding site, shifting Thr⁹⁷ and Gly⁹⁶ toward the inhibitor molecule. We conclude that the decreased inhibitory potency observed for glyphosate is a result of these mutation-induced long-range structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.

Received for publication, July 9, 2007 , and in revised form, September 11, 2007.

The atomic coordinates and structure factors (code 2qfs, 2qft, 2qfq, and 2qfu) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant 1R01 GM70633-02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: Drug Discovery Program, H. Lee Moffitt Cancer Research Inst., Tampa, FL 33612. Tel.: 813-745-4703; Fax: 813-745-6748; E-mail: ernst.schonbrunn{at}moffitt.org.

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				T. Funke, Y. Yang, H. Han, M. Healy-Fried, S. Olesen, A. Becker, and E. Schonbrunn Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 -> Ile and Pro101 -> Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli J. Biol. Chem., April 10, 2009; 284(15): 9854 - 9860. [Abstract] [Full Text] [PDF]