Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

• Published in: Biotechnology journal 2024-03, Vol.19 (3), p.e2300637-n/a
• Main Authors: Dai, Chen, Tian, Jia‐Xin, Chen, Yu‐Feng, Ni, Yue‐Han, Cui, Lei, Cao, Hai‐Xing, Song, Lin‐Lin, Xu, Shen‐Yuan, Wang, Ya‐Jun, Zheng, Yu‐Guo
• Format: Article
• Language: English
• Subjects: Aldehyde Reductase; Aldo-Keto Reductases - chemistry; Aldo-Keto Reductases - genetics; aldo‐keto reductase; Caproates - chemistry; computer‐aided design; organic solvent tolerance; thermostability
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.202300637

The aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs. Graphical and Lay Summary Naturally evolved enzymes are frequently inactivated under harsh industrial conditions like high temperature and organic solvents; therefore, it is crucial to enhance protein stability for biocatalytic process. A computer‐assisted strategy was used to improve the stability of the aldo‐keto reductase from Kluyveromyces dobzhanskii. The modified variant KdAKRM10 has industrial application value in the asymmetric synthesis of t‐butyl 6‐chloro‐(3R, 5S)‐dihydroxyhexanoate, which is the key chiral building block of the lipid‐lowering drug rosuvastatin.