Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5enolpyruvyishikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA(A.sp). A phylogenetic analysis revealed that AroA(A.sp) (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K-M for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA(A.sp) were 78 mu M and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type Aro(A.sp). The specific activity of the wild-type AroA(A.sp), the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. (C) 2014 Published by Elsevier Inc.