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

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Synthesis

Background:

• Short-chain dehydrogenases/reductases (SDRs) are valuable biocatalysts for chiral alcohol synthesis.
• Limited substrate specificity of wild-type SDRs restricts their application in synthesizing non-native compounds.

Purpose of the Study:

• To engineer the Serratia marcescens SDR (SmSDR) for enhanced catalytic performance towards 2-chloro-1-(3-hydroxyphenyl) ethan-1-one (CHL).
• To develop a computational-experimental framework for overcoming substrate limitations in SDR enzymes for drug intermediate synthesis.

Main Methods:

• Rational enzyme engineering guided by sequence conservation, active site mapping, and network modeling.
• Construction and biochemical assay of 14 SmSDR variants.
• Molecular dynamics simulations and MM-PBSA analysis to investigate binding interactions and stability.

Main Results:

• Engineered SmSDR variants achieved >70% conversion of CHL, significantly outperforming the wild-type enzyme (~28% conversion).
• Computational analyses confirmed improved substrate binding and retention within the engineered active sites.
• Engineered variants exhibited up to -10 kcal·mol^-1 improvement in binding free energy compared to wild type.

Conclusions:

• A robust computational-experimental workflow was established for tailoring SDRs towards specific non-native substrates.
• The engineered SmSDR provides a promising biocatalyst for the efficient asymmetric synthesis of (R)-Phenylephrine intermediates.
• This approach offers a framework for advancing selective biocatalysis in pharmaceutical manufacturing.