Carbonyl reductases (CRs) are important biocatalysts that catalyze the asymmetric reduction of carbonyl compounds to afford optically active alcohols.We have been studying a nicotinamide-adenine dinucleotide (NAD)-dependent carbonyl reductase from Gluconobacter oxydans (GoCR), which could reduce different kinds of ketone substrates to corresponding alcohols with moderate to high enantioselectivity.To understand the differential enantiopreference from the point of view of tertiary structure and thus to engineer the enantioselectivity of GoCR to afford desirable products, the crystal structure of GoCR was firstly determined, docking and molecular dynamics (MD) simulations were then performed using selected substrates.A mathematic model based on the (anti)Prelogs rule was constructed to help us monitor the substrates orientations in the MD process.For the ketone substrates tested here,these computational studies well explained the experimental enantiopreference data and helped us understand the enantioselectivity mechanism at the molecular level.For the reduction of 2-oxo-4-phenylbutyrate (OPBE), three binding site residues, Trp193, Tyr149, and Cys93 were predicted to be the key points that may exert significant influence on the product selectivity.Through site-directed mutations, single-point mutant W193A was constructed and proved to reduce OPBE to ethyl (R)-2-hydroxy-4-phenylbutyrate (R-HPBE) with a dramatic improved ee (>99%, R) compared to wild type (43.2 %, R).Meanwhile, double mutant Y149A/C93V was proved to even invert the ee of wild type to 79% (S).