Regulating the stereoselectivity of medium-chain dehydrogenase/reductase by creating an additional active pocket accommodating prochiral heterocyclic ketones

Medium chain dehydrogenase/reductase (MDR) is a robust catalyst for the asymmetric synthesis of chiral heterocyclic alcohols, essential building blocks in pharmaceuticals. However, the regulatory mechanism of stereoselective complementary reduction of heterocyclic ketones by carbonyl reductase (CR) is unclear. Structure-guided creation of an additional substrate-binding active pocket inversed the stereoselectivity of SpCR from Spathaspora passalidarum. The mutant m48 showed improved catalytic activity towards the 12 tested heterocyclic ketones (conversion rate >99%, ee value > 99%). Hence, we regulated the stereoselectivity of MDR by creating an active pocket suitable for substrate localisation. This strategy has a guiding significance in addition to the conventional method for stereoselectivity modification of MDR.