Integrated engineering at distal site and active center regulates stereoselectivity and activity of carbonyl reductase towards N-Boc-pyrrolidone

Green and efficient biosynthesis of two enantiomeric products of N-Boc-pyrrolidinol (NPBL) as key pharmaceutical blocks for treatment of cancer and HIV has received much attention. The precursor, N-Boc-pyrrolidone (NPBO), is considered as a difficult-to-reduce ketone by carbonyl reductases, because it has a bulky Boc group and sterically similar substitutions on either side of the carbonyl group resulting in low stereoselectivity and conversion. Moreover, activity enhancement concomitant with reversal of stereoselectivity is challenging. Carbonyl reductase (CpCR) from Candida parapsilosis shows 90 %ee for (S)-NBPL, but low catalytic efficiency. In this study, we considered both activity and selectivity, and proposed an integrated engineering strategy. The distal site was introduced from an activity perspective and the active pocket sites were introduced by virtual saturation mutagenesis from a stereoselectivity perspective. Mutants L34A/W116A (95 %ee (S)) and L34A/W116T/F285C/W286S (94 %ee (R)) were obtained with 26.8-fold and 2.9-fold higher catalytic efficiencies, respectively. Molecular dynamics simulations revealed the mechanism of stereoselective flip-flop and activity enhancement; mutation altered the substrate-binding mode and changed the shape and size of the cavities, thus contributing to the change in the ratio of active conformations in the pre-reactive state. This study, which differs from the traditional method of exchanging large and small pockets, provides a viable approach for the rational design of carbonyl reductases with high stereoselectivity for target substrates.