Construction of the chiral 24R-OH squalamine intermediate by an engineered ketoreductase from Rhodococcus sp

The 24R-OH construction remains a bottleneck in squalamine synthesis. In this study, a ketoreductase from Rhodococcus sp. was screened out, which could catalyze the conversion of 7α-hydroxy-5α-cholestan-3, 24-dione to the key intermediate (7α, 24R)-dihydroxy-5α-cholestan-3-one (24R-OH). A mechanism-driven computational design strategy was employed, and regional engineering targeting the substrate channel (D42A), flexible regions (P89G, P158S), and a structural stabilization zone (A327P) was conducted to generate the quadruple mutant D42A/P89G/P158S/A327P. Under the reaction conditions at 10 mg/mL substrate and 25% (v/v) isopropanol, the mutant achieved a conversion of 99.1% with a diastereomeric excess of >99.9%. Further investigation through molecular dynamics simulations, free energy calculations, and hydrogen-bonding network analysis revealed that the mutation facilitated substrate access and product release while stabilizing the protein structure. This study provides a robust and industrially viable biosynthetic route for efficiently producing 24R-OH, the key intermediate in the squalamine synthesis.