Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 67, Issue 6, Pages 1405-+Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.67.1405
Keywords
bacterial aroA; glyphosate; mutagenesis; herbicide; 5-enolpyruvylshkimate-3-phosphate synthase (EPSPS)
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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.
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