Site-Directed Mutagenesis of Putative Active Site Residues of 5-Enolpyruvylshikimate-3-phosphate Synthase

Abstract

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

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

“…Two additional interactions between substrate and inhibitor are mediated through the side chain of Lys-22 and water molecule W2. The glyphosate binding site is dominated by charged residues from both domains of the enzyme, of which Lys-22, Arg-124, and Lys-411 have previously been implicated in PEP binding (27). In the absence of glyphosate from otherwise identical crystallization conditions (see Methods), a phosphate and a formate ion (Fig.…”

A T42M Substitution in Bacterial 5-Enolpyruvylshikimate-3-phosphate Synthase (EPSPS) Generates Enzymes with Increased Resistance to Glyphosate

“…For example, mutations of R100A, D242A, and D384A show a drastic decrease in activity but none of these amino acids are involved in substrate binding, and instead may hinder domain closure. 15) Simarily, we speculate that the T42M amino acid substitution alters the relationship of the two globular domains.…”

Chemical shift mapping of shikimate‐3‐phosphate binding to the isolated N‐terminal domain of 5‐enolpyruvylshikimate‐3‐phosphate synthase

“…The secondary structure prediction of these chemical shift changes is shown and compared with the secondary structure of the N-terminal domain within the full-length EPSP synthase as determined by X-ray crystallography [5]. mutagenesis (unpublished results and [15]), with 10% and undetectable residual activity, respectively, for the alanine substitutions. Ser23 is involved in S3P binding only in the Scho « nbrunn binary complex structure [7], while Arg27 and Ser197 are implicated in both binary complex structures.…”