Synthesis of a chiral alcohol using a rationally designed Saccharomyces cerevisiae reductase and a NADH cofactor regeneration system

Abstract

Abstract Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs that function as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Yeast reductase YDL124W has been shown to convert ethyl 4-chloro-3-oxo butanoate (ECOB) into (S)-ECHB in an enantioselective manner. In that reduction, YDL124W absolutely prefers NADPH as a cofactor to NADH. NADPH is, however, much more expensive and unstable than NADH. In this study, four amino acid residues that directly interact with the 2′-phosphate group of adenosine ribose of NADPH were identified based on the homology model and docking model of YDL124W with ECOB and NADPH. Among various single mutants altered in these target residues, R264H evidenced noticeably increased reductase activity with NADH. The double mutant, R264H/R27Y, had more profoundly enhanced reductase activity with NADH. Kinetic analysis results demonstrated that these mutants had reduced Km values toward NADH and increased kcat/Km values in comparison with the wild-type. Computer modeling showed that no hydrogen bond could be formed between the wild-type and 2′-hydroxyl group of adenosine ribose of the NADH cofactor, whereas three hydrogen bonds and one π–π stacking interaction could be formed between R264H or R264H/R27Y mutants and the NADH cofactor. These two mutants could efficiently produce (S)-ECHB using NADH as a cofactor in conjunction with formate dehydrogenase as a coupling enzyme.