Semirational engineering of an aldo–keto reductase KmAKR for overcoming trade‐offs between catalytic activity and thermostability

Abstract

Enzyme engineering usually generates trade‐offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo–keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), and t‐butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer‐aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the “best” mutant KmAKRM13(W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9, respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9. Structural analysis indicated that stabilization of the α‐helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400\u2009g/l t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) in 8.0\u2009h at a high substrate to catalyst ratio (S/C) of 106.7\u2009g/g, giving diastereomerically pure (3R,5S)‐CDHH (>99.5% d.e.P) with a space‐time yield (STY) of 449.2\u2009g/l·d.