Institution: | 1. College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology, Beijing, China
Contribution: Formal analysis (lead), Methodology (lead);2. College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology, Beijing, China
Contribution: Formal analysis (equal), Methodology (equal);3. College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology, Beijing, China
Contribution: Data curation (equal);4. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Contribution: ?Investigation (lead);5. College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China;6. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China |
Abstract: | R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from ?17.0 kJ/mol to ?29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales. |