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

Abstract

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...

Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

“…mutants at G101 or T102) have greatly increased K m values (decreased affinity) for PEP when expressed in Escherichia coli (Eschenburg et al, 2002;Funke et al, 2009; for review, see Sammons and Gaines, 2014). In contrast, P106 substitutions confer weak glyphosate resistance but preserve adequate EPSPS functionality (Healy-Fried et al, 2007; for review, see Sammons and Gaines, 2014). Aside from P106 EPSPS gene mutations, there are other glyphosate resistance mechanisms, including EPSPS gene amplification and nontarget-site reduced glyphosate translocation/nontarget-site increased vacuole sequestration (Lorraine-Colwill et al, 2002;Gaines et al, 2010;Ge et al, 2010; for review, see Powles and Preston, 2006;Shaner, 2009;Powles and Yu, 2010;Sammons and Gaines, 2014).…”

Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli

“…7). In the WT and P101S enzymes, the hydroxyl group of Thr 97 is hydrogen-bonded with the side chain of Asn 26 ( Fig. 8).…”

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

“…Numerous amino acid residues that affect EPSPS glyphosate resistance have already been identified (1,8,10,13,15,19,20,24,25,31,35,40,50). Notably, these residues are mostly distributed in two regions according to their 3D structure.…”