Engineering a Carbonyl Reductase to Simultaneously Increase Activity Toward Bulky Ketone and Isopropanol for Dynamic Kinetic Asymmetric Reduction via Enzymatic Hydrogen Transfer

It is very challenging to engineer a carbonyl reductase with simultaneously improved activity toward both a bulky ketone and isopropanol, enabling substrate-coupled NAD(P)H regeneration for the ketone reduction. In this study, a mutant M242F/Q245T of the carbonyl reductase from Sporobolomyces salmonicolor was identified to simultaneously enhance its activity toward both methyl 2-phthalimidomethyl-3-oxobutanoate (1a) and isopropanol and catalyze the dynamic kinetic asymmetric reduction of 1a to a single stereoisomer of the product by hydrogen transfer from isopropanol. Crystal structural and computational analysis suggested that it might be due to the synergic effect of 242F and 245T by reshaping the substrate entrance tunnel and enzyme active site. This study unprecedentedly showed that a carbonyl reductase could be engineered to increase its activity toward both bulky ketones and isopropanol, and the target chiral alcohol can be efficiently prepared via substrate-coupled cofactor regeneration.