Authors
Shu-Fang Li, Ting Zou, Sen-Yu Fu, Jin-Chuan Li, Yuan Li, Ya-Ru Shen, Ya-Ping Xue, Yu-Guo Zheng
Published in
Biotechnology and bioengineering. Oct 06, 2026. Epub Oct 06, 2026.
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 (Tm), a 2.3-fold prolongation of half-life (t1/2) at 40°C (reaching 75.3 h), a 3.14-fold improvement in catalytic efficiency (kcat/Km) 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.
PMID:
42834806
Bibliographic data and abstract were imported from PubMed on 06 Oct 2026.
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