title: 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 K(m)[PEP] and a 21- to 26-fold increased K(i)[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.
authors.affiliation: Biotechnology Research Center, The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou 510275, The People's Republic of China.
authors.forename: Ming
authors.initials: M
authors.lastname: He
authors.forename: Yan-Fang
authors.initials: YF
authors.lastname: Nie
authors.forename: Peilin
authors.initials: P
authors.lastname: Xu
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004351:Drug Resistance / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006540:Herbicides / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D030821:Plants, Genetically Modified / Q000201:enzymology; D012486:Salmonella typhimurium / Q000201:enzymology; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate