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 T-50(15) and T-m values were increased by 10.2 and 10.0 degrees C, respectively, and half-life (t(1/2)) at 40 degrees 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.