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

• DOI: 10.1002/bit.27913
• Publication Date: 2021-08-10T16:03:00Z
• Authors: Shu‐Fang Li; Jian‐Yong Xie; Shuai Qiu; Shen‐Yuan Xu; Feng Cheng; Ya‐Jun Wang; Yu‐Guo Zheng
• Keywords: Thermostability; Protein Engineering

Abstract Enzyme engineering usually generates trade‐offs between activity, stability, and selectivity. Herein, we report semirational of an aldo–keto reductase (AKR) Km AKR for simultaneously enhancing its thermostability catalytic activity. Previously, constructed 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 )‐CDHH), 6‐cyano‐(3 )‐dihydroxyhexanoate, the key chiral building blocks rosuvastatin atorvastatin. Under guidance computer‐aided design including consensus residues analysis molecular dynamics (MD) simulations, K164, S182, S232, Q266 were dug out their conferring roles, generating “best” mutant M13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The T m 50 15 values 10.4 6.1°C higher than that , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over . Structural indicated stabilization α ‐helixes mainly contributed to enhancement. optimized conditions, completely asymmetrically reduced 400 g/l 6‐chloro‐(5 )‐hydroxy‐3‐oxohexanoate ((5 )‐CHOH) in 8.0 h at high substrate catalyst ratio (S/C) 106.7 g/g, giving diastereomerically pure (3 )‐CDHH (>99.5% d.e P ) with space‐time yield (STY) 449.2 g/l·d.