Structure-Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion.
Chemphyschem
; 25(11): e202400008, 2024 Jun 03.
Article
em En
| MEDLINE
| ID: mdl-38514394
ABSTRACT
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 46.19â
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 0.08â
U/g. By introducing additional beneficial mutations, MT11 was constructed with significantly increased specific activity of 2.29â
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.
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Base de dados:
MEDLINE
Assunto principal:
Oxigenases
/
Omeprazol
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China