Structure-guided evolution of cyclohexanone monooxygenase toward bulky omeprazole sulfide: substrate migration and stereoselectivity inversion.

Structure-guided engineering of a CHMO from Amycolatopsis methanolica (AmCHMO) 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 AmCHMO, while reshaping of substrate binding pocket is important for stereoselective inversion.Based on MD simulation,  pre-reaction states of MT3-OPSproR, MT8-OPSproS, and MT11-OPSproS 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.