Engineering of Cyclohexanone Monooxygenase for the Enantioselective Synthesis of ( S )-Omeprazole

作者：Yan Zhang, Yinqi Wu, Na Xu, Qian Zhao, Hui‐Lei Yu, Jian‐He Xu · 发表于：ACS Sustainable Chemistry & Engineering · 年份：2019 · DOI：10.1021/acssuschemeng.9b00224 · 被引用次数：68 · 研究领域：Enzyme Catalysis and Immobilization、Enzyme function and inhibition、Steroid Chemistry and Biochemistry

Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO ( Ac CHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of Ac CHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of Ac CHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/g prot ). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate ...