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

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 T₅₀ 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, 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.