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 reductasewith simultaneouslyimproved activity toward both a bulky ketone and isopropanol, enablingsubstrate-coupled NAD(P)H regeneration for the ketone reduction. Inthis study, a mutant M242F/Q245T of the carbonyl reductase from Sporobolomyces salmonicolor was identified to simultaneouslyenhance its activity toward both methyl 2-phthalimidomethyl-3-oxobutanoate(1a) and isopropanol and catalyze the dynamic kineticasymmetric reduction of 1a to a single stereoisomer ofthe product by hydrogen transfer from isopropanol. Crystal structuraland computational analysis suggested that it might be due to the synergiceffect of 242F and 245T by reshaping the substrate entrance tunneland enzyme active site. This study unprecedentedly showed that a carbonylreductase could be engineered to increase its activity toward bothbulky ketones and isopropanol, and the target chiral alcohol can beefficiently prepared via substrate-coupled cofactor regeneration.