Chemo-Enzymatic Synthesis of a Squalamine Intermediate by an Engineered Ketoreductase from Novosphingobium aromaticivorans

7α, 24R-Dihydroxy-5α-cholestan-3-one serves as a key intermediate in the synthesis of squalamine. In this study, a simple and efficient three-step chemo-enzymatic method was developed with a total yield of 61% by utilizing an engineered ketoreductase from Novosphingobium aromaticivorans (NaKRED). NaKRED was a NADH-dependent short-chain alcohol dehydrogenase, and the mutant variant (Y199A/N149L) exhibited a 94.3% conversion rate of substrate 1a and 99.8% diastereoselectivity. At the optimized reaction system, the substrate concentration at 10 mM afforded a product yield of 51.2% and a titer of 2.153 g/L while maintaining stereoselectivity above 99.8%. Crystal structures of the NAD+-free and NAD+-bound wild-type and Y199A/N149L mutant revealed that the intrinsically Ser-Tyr-Lys catalytic triad machinery formed a prominent and flexible ammonium cation hole into which the NADH molecule was buried. NADH featured two different conformations in tetrameric and octameric ketoreductases, and mediated the catalytic activity by undergoing a conformational change to form a productive substrate binding conformation (A-form) based on the crystal structure and molecular dynamics simulation. Our research presented not only an effective strategy to produce the key squalamine intermediate but also revealed a previously unrecognized NADH-regulated mechanism in KRED-catalyzed ketone reduction.