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

Abstract The aldo‐keto reductase (AKR) Kd AKR from Kluyvermyces dobzhanskii can reduce t ‐butyl 6‐chloro‐(5 S )‐hydroxy‐3‐oxohexanoate ((5 S )‐CHOH) to t ‐butyl 6‐chloro‐(3 R ,5 S )‐dihydroxyhexanoate ((3 R ,5 S )‐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 Kd AKR M6 . Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant Kd AKR M10 ( Kd AKR M6 ‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T 50 15 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, Kd AKR M10 demonstrated improved resistance to organic solvents compared to that of Kd AKR M6 . Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of Kd AKR M10 , thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.