Controlling Stereopreferences of Carbonyl Reductases for Enantioselective Synthesis of Atorvastatin Precursor

Carbonyl reductase (CR)-catalyzed asymmetric reduction offers an approach for producing t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b), which serves as a key building block in atorvastatin (Liptor). However, controlling the stereopreference of CR with the desired selectivity remains challenging because natural CRs usually exhibit Prelog preference. Moreover, transferring knowledge from engineered anti-Prelog CRs to other CRs is difficult. Herein, the key residues that regulate the stereopreference of a CR from Kluyveromyces marxianus (KmCR) were identified by a semirational engineering toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1a). A structural switch that consists of the key residues was discovered, and related structural features were summarized to predict the stereopreference by analyzing the structural information and multiple-sequence alignment of structure-available CRs carefully. According to the obtained knowledge, the simultaneous mutation of four key residues enabled the conversion of Prelog-selectivity of KmCR into a complete anti-Prelog selectivity (de(p) > 99% (R) for (3R,5R)-1b). Moreover, the stereopreferences of 11 CRs that share 20-40% sequence identities with KmCR were predicted successfully and engineered experimentally. The knowledge gained from this protein engineering study on KmCR has universal significance for CRs toward (5R)-1a.

Automated summary

Key residues regulating the stereopreference of a carbonyl reductase from Kluyveromyces marxianus (KmCR) were identified through semi-rational engineering, leading to the successful conversion of Prelog selectivity to complete anti-Prelog selectivity. This knowledge was then used to successfully predict and engineer the stereopreferences of other CRs sharing sequence identities with KmCR.