Engineering the α‐Helix Dynamics of Hydroxysteroid Dehydrogenase by Computational Design to Balance Catalytic Activity and Stability

ABSTRACT The stability‐activity trade‐off remains a major bottleneck in engineering robust biocatalysts for industrial applications. Here, we developed an integrated computational strategy to simultaneously enhance the catalytic activity and thermostability of 7α‐hydroxysteroid dehydrogenase (7α‐HSDH) by engineering highly flexible regions, with a particular focus on α‐helices. Evolutionary conservation analysis combined with multi‐temperature molecular dynamics (MD) simulations was first applied to identify weakly conserved and flexible α‐helices. Subsequently, computational tools (PROSS and Pythia) were utilized to generate mutations within these flexible regions. Through modular assembly of synergistic mutations, the optimal mutant M9 (D12K/V15I/V50E/I56R/D66A/T76R/A126I/A132H/S158A/S161A/A203P/H208Q) was obtained. Compared with the WT, M9 exhibited a 10.5°C increase in melting temperature ( T m ), a 2.3‐fold prolongation of half‐life ( t 1/2 ) at 40°C (reaching 75.3 h), a 3.14‐fold improvement in catalytic efficiency ( k cat /K m ) to 29,032.73 mM −1 s −1 , and enhanced substrate tolerance. Structural and molecular dynamics analyses indicated that the enhanced performance of M9 was associated with increased rigidity of key α‐helix regions, redistribution of surface electrostatic potential, and remodeling of the substrate‐binding pocket. This study thus offers a novel design framework for improving the catalytic activity, thermostability, and substrate tolerance, balancing the stability‐activity trade‐off.