Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site
1991
Padgette, S.R. | Re, D.B. | Gasser, C.S. | Eichholtz, D.A. | Frazier, R.B. | Hironaka, C.M. | Levine, E.B. | Shah, D.M. | Fraley, R.T. | Kishore, G.M.
The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app)(glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.
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