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) Km AKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed Km AKR M9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t ‐butyl 6‐chloro‐(3 R ,5 S )‐dihydroxyhexanoate ((3 R ,5 S )‐CDHH), and t ‐butyl 6‐cyano‐(3 R ,5 R )‐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 Km AKR M13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The T m and T 50 15 values of Km AKR M13 were 10.4 and 6.1°C higher than that of Km AKR M9 , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over Km AKR M9 . Structural analysis indicated that stabilization of the α ‐helixes mainly contributed to thermostability enhancement. Under the optimized conditions, Km AKR M13 completely asymmetrically reduced 400 g/l t ‐butyl 6‐chloro‐(5 S )‐hydroxy‐3‐oxohexanoate ((5 S )‐CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3 R ,5 S )‐CDHH (>99.5% d.e . P ) with a space‐time yield (STY) of 449.2 g/l·d.