A t42m substitution in bacterial 5-enolpyruvylshikimate-3-phosphate synthase (epsps) generates enzymes with increased resistance to glyphosate

Abstract

Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased Km[PEP] and a 21- to 26-fold increased Ki[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design. © 2003 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.