文献类型：期刊文献

英文题名：Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance

作者：Ren, Shi-Miao[1];Liu, Feng[1];Wu, Yin-Qi[1];Chen, Qi[1];Zhang, Zhi-jun[1];Yu, Hui-Lei[1];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2021

卷号：118

期号：2

起止页码：737

外文期刊名：BIOTECHNOLOGY AND BIOENGINEERING

收录：;EI(收录号：20204809554930);WOS:【SCI-EXPANDED(收录号:WOS:000586207500001)】；

基金：Fundamental Research Funds for the Central Universities, Grant/Award Number: 22221818014; National Natural Science Foundation of China, Grant/Award Numbers: 21922804, 21672063; National Key Research and Development Program of China, Grant/Award Number: 2019YFA09005000; Fundamental Research Funds for the Central Universities, Grant/Award Number: 22221818014

语种：英文

外文关键词：biocatalysis; protein engineering; (S)‐ omeprazole; thioether monooxygenases

摘要：AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and alpha-helical domain based on structural characteristics of AcCHMO. 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. AcCHMOM7 (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 mu M. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.