From structure to function: Computational engineering of SmSDR for enhanced activity

Short-chain dehydrogenases/reductases (SDRs) are promising biocatalysts for the asymmetric synthesis of chiral alcohols, yet their limited activity toward non-native substrates constrains broader application. In this study, the Serratia marcescens SDR (SmSDR) was rationally engineered to enhance catalytic performance toward 2-chloro-1-(3-hydroxyphenyl) ethan-1-one (CHL), a key intermediate in (R)-Phenylephrine synthesis. Sequence conservation, active site mapping, tunnel analysis, and residue interaction network modelling guided the design of 14 variants targeting cofactor orientation, substrate gating, and structural dynamics. Biochemical assays demonstrated that while the wild-type enzyme achieved only ~28 % conversion of CHL, several engineered variants exceeded 70 % conversion. Molecular dynamics simulations confirmed improved substrate retention in remodeled active sites, and MM-PBSA analysis indicated more favorable binding free energies, with improvements of up to -10 kcal·mol^-1 relative to wild type. These results establish a robust computational-experimental workflow for overcoming substrate-specific limitations of SDRs and provide a framework for tailoring enzymes for selective biocatalysis in drug intermediate synthesis.