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Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance
Author(s) -
Ren ShiMiao,
Liu Feng,
Wu YinQi,
Chen Qi,
Zhang Zhijun,
Yu HuiLei,
Xu JianHe
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27604
Subject(s) - mutagenesis , mutant , thioether , chemistry , monooxygenase , cyclohexanone , substrate (aquarium) , phenocopy , sulfoxide , stereochemistry , combinatorial chemistry , site directed mutagenesis , acinetobacter calcoaceticus , biochemistry , catalysis , enzyme , biology , organic chemistry , acinetobacter , cytochrome p450 , gene , ecology , antibiotics
Ac CHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus , is a typical Type I Baeyer–Villiger monooxygenase (BVMO). We previously obtained the Ac CHMO M6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the Ac CHMO M6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD‐binding domain, NADPH‐binding domain, and α ‐helical domain based on structural characteristics of Ac CHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. Ac CHMO M7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the K i of the substrate OPS from 34 to 265 μM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of Ac CHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.