Design of a Distal Site Saturation Test-Iterative Parallel Mutagenesis for Engineering Hydroxysteroid Dehydrogenase

Distal protein engineering facilitates the efficient identification of novel modification sites and synergistic modulation of enzyme functions to meet the demands of biocatalysts for industrial applications. Using hydroxysteroid dehydrogenase as a target protein, this study presents a distal site saturation test-iterative parallel mutagenesis (DSST-IPM) strategy to design high-performance enzymes. Twelve single-point mutations were identified to improve the stability–activity trade-off in the distal site, targeting 34 residues. S176G and Q245L exhibited a significant melting temperature (Tm) increase of 11.3 and 10.6 °C, respectively. Iterative parallel screening of mutations yielded the mutation7β-HSDH-M6b, which showed a 13.3 °C higher Tm and 5.92-fold higher catalytic activity (kcat/Km) than the wild-type 7β-HSDH. Systematic analysis of molecular dynamics simulations, quantum mechanical calculations, and dynamic cross-correlation matrix (DCCM), the mechanism behind the enhanced catalytic performance of M6b was elucidated. It uncovered that the critical fourth shell could influence conformational dynamics during the enzyme-catalyzed reaction, leading to alterations in the inter-regional force interaction network. This study thus offers an advanced design framework for improving the efficiency of engineering highly active, thermostable biocatalysts for industrial applications.