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Site‐directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism
Author(s) -
Pan Haifeng,
Xie Zhipeng,
Bao Wenna,
Cheng Yongqing,
Zhang Jianguo,
Li Yongquan
Publication year - 2011
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2011.07.006
Subject(s) - chemistry , stereochemistry , active site , alanine , hydrolase , site directed mutagenesis , stereospecificity , epoxide hydrolase , enzyme , dehalogenase , biochemistry , amino acid , hydrolysis , mutagenesis , catalysis , mutant , gene , microsome
Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis ‐epoxysuccinate to L(+)‐tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16–23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two‐step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.