Structure‐Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion

Abstract Structure‐guided engineering of a CHMO from Amycolatopsis methanolica ( Am CHMO) was performed for asymmetric sulfoxidation activity and stereoselectivity toward omeprazole sulfide. Initially, combinatorial active‐site saturation test (CASTing) and iteratively saturation mutagenesis (ISM) were performed on 5 residues at the “bottleneck” of substrate tunnel, and MT3 was successfully obtained with a specific activity of 46.19 U/g and R ‐stereoselectivity of 99 % toward OPS. Then, 4 key mutations affecting the stereoselectivity were identified through multiple rounds of ISM on residues at the substrate binding pocket region, resulting MT8 with an inversed stereoselectivity from 99 % ( R ) to 97 % ( S ). MT8 has a greatly compromised specific activity of 0.08 U/g. By introducing additional beneficial mutations, MT11 was constructed with significantly increased specific activity of 2.29 U/g and stereoselectivity of 97 % ( S ). Enlarged substrate tunnel is critical to the expanded substrate spectrum of Am CHMO, while reshaping of substrate binding pocket is important for stereoselective inversion. Based on MD simulation, pre‐reaction states of MT3‐OPS proR , MT8‐OPS proS , and MT11‐OPS proS were calculated to be 45.56 %, 17.94 %, and 28.65 % respectively, which further confirm the experimental data on activity and stereoselectivity. Our results pave the way for engineering distinct activity and stereoselectivity of BVMOs toward bulky prazole thioethers.