Semirational engineering of an aldo–keto reductase KmAKR for overcoming trade‐offs between catalytic activity and thermostability. Issue 11 (16th August 2021)

• Record Type: Journal Article
• Authors: Li, Shu‐Fang; Xie, Jian‐Yong; Qiu, Shuai; Xu, Shen‐Yuan; Cheng, Feng; Wang, Ya‐Jun; Zheng, Yu‐Guo
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 11(2021)
• Page Start: 4441
• Page End: 4452
• Publication Date: 2021-08-16
• Subjects: aldo–keto reductase -- computer‐aided design -- protein engineering -- thermostability; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27913
• Languages: English
• ISSNs: 0006-3592

Abstract

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 AKRM9 (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 AKRM13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The T m and T 50 15 values of Km AKRM13 were 10.4 and 6.1°C higher than that of Km AKRM9, respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over Km AKRM9 . Structural analysis indicated that stabilization of the α ‐helixes mainly contributed to thermostability enhancement. Under the optimized conditions, Km AKRM13 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. Graphical abstract: The thermostability of an aldo–keto reductase Km AKR was improved with simultaneously enhancing catalytic activity through a computer‐aided design strategy that includes consensus residues analysis and molecular dynamics (MD) simulations. The "best" mutant of Km AKR also displayed significantly elevated organic solvent tolerance and substrate tolerance, displaying great potentials in industrial applications. The successful engineering strategy provides insights into the coevolution of activity and thermostability of aldo–keto reductases.