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

Abstract

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 KdAKR_M6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKR_M10 (KdAKR_M6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T₅₀¹⁵ and T_m values were increased by 10.2 and 10.0°C, respectively, and half‐life (t_1/2) at 40°C was increased by 17.6 h. Additionally, KdAKR_M10 demonstrated improved resistance to organic solvents compared to that of KdAKR_M6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKR_M10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.