Engineering of a Baeyer-Villiger monooxygenase reveals key residues for the asymmetric oxidation of omeprazole sulfide.

The structure-guided engineering of a BVMO from Rhodococcus aetherivorans ( Ra BVMO) was performed for its asymmetric sulfoxidation activity toward omeprazole sulfide. Based on the structural model of Ra BVMO, key residues that line the substrate entrance tunnel and the binding pocket were identified, and variants were interrogated with sulfides of varied sizes. The best mutant MT2 (F442A/R337P) was obtained with a specific activity of 2.54 U g -1 and excellent enantioselectivity (≥99%, S ) toward omeprazole sulfide, while wild-type Ra BVMO exhibited no activity. Further structural analysis reveals that both mutations, F442A and R337P, could render an expanded substrate tunnel and an enlarged substrate binding pocket to enable easier access to the catalytic center for omeprazole sulfide. This work provides valuable guidance for engineering-related BVMOs for improved activity and enantio-preference toward bulky substrates.