文献类型：期刊文献

英文题名：Regioselectivity Engineering of Epoxide Hydrolase: Near-Perfect Enantioconvergence through a Single Site Mutation

作者：Li, Fu-Long[1];Kong, Xu-Dong[1,2];Chen, Qi[1];Zheng, Yu-Cong[1];Xu, Qin[3,4];Chen, Fei-Fei[1];Fan, Li-Qiang[1];Lin, Guo-Qiang[2];Zhou, Jiahai[2];Yu, Hui-Lei[1];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai Collaborat Innovat Ctr Biomfg Technol, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Ctr Excellence Mol Synth, Shanghai Inst Organ Chem, Shanghai 200032, Peoples R China;[3]Shanghai Jiao Tong Univ, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China;[4]Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China

年份：2018

卷号：8

期号：9

起止页码：8314

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20183505763344);WOS:【SCI-EXPANDED(收录号:WOS:000444364800060)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21536004, 21672063, 31770772), Shanghai Commission of Science and Technology (No. 15JC1400403), the Strategic Priority Research Program (B) of the CAS (XDB20000000), the Fundamental Research Funds for the Central Universities (No. 22221818014) and an Open Fund of the State Key Laboratory of Bioorganic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences. We are very grateful for the access to beamline BL17U1 and BL19U1 at Shanghai Synchrotron Radiation Facility and thank the beamline staff for technical help. We thank Simon Partridge, PhD, from Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript.

语种：英文

外文关键词：biocatalysis; epoxide hydrolase; crystal structure; regioselectivity; enantioconvergence; protein engineering

摘要：An epoxide hydrolase from Vigna radiata (VrEH2) affords partial enantioconvergence (84% ee) in the enzymatic hydrolysis of racemic p-nitrostyrene oxide (pNSO), mainly due to insufficient regioselectivity for the (S)-enantiomer (r(s) = alpha(s)/beta(s) = 7.3). To improve the (S)-pNSO regioselectivity, a small but smart library of VrEH2 mutants was constructed by substituting each of four key residues lining the substrate binding site with a simplified amino acid alphabet of Val, Asn, Phe, and Trp. Among the mutants, M263N attacked almost exclusively at C alpha in the (S)-epoxide ring with satisfactory regioselectivity (r(s) = 99.0), without compromising the original high regioselectivity for the (R)-epoxide (r(R) = 99.0), resulting in near-perfect enantioconvergence (>99% analytical yield, 98% ee). Structural and conformational analysis showed that the introduced Asn263 formed additional hydrogen bonds with the nitro group in substrate, causing a shift in the substrate binding pose. This shift increased the difference in attacking distances between C alpha and C beta, leading to an improved regiopreference toward (S)-pNSO and affording near-perfect enantioconvergence.