Abstract：
Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.
摘要：
羟基类固醇脱氢酶（HSDH）对于胆汁酸代谢至关重要，并影响胆汁酸池的大小和肠道微生物群组成。具有高活性的HSDHs，热稳定性,和底物选择性是构建用于疾病治疗的工程细菌的基础。在这项研究中,我们设计了涉及HSDHSt-2-2的Thr15和Arg16残基的辅因子结合位点的突变。T15A,R16A,R16Q突变体表现出7.85-，2.50-，催化活性比野生型高4.35倍，分别,同时还显示出改变的底物偏好（从牛磺胆酸（TCA）到牛磺鹅去氧胆酸（TCDCA））。这些突变体显示出较低的Km和较高的kcat值，表明与底物的结合更强，导致3190-，3123-,TCDCA氧化的kcat/Km值高3093倍。此外，T15A的Tm值,R16A,发现R16Q突变体增加了4.3°C，6.0°C,和7.0°C，分别。分子结构分析表明，内部氢的重塑和表面突变可以提高催化活性和热稳定性，并改变了催化三联体之间的相互作用，辅因子结合位点，和底物可以改变底物偏好。这项工作为通过靶向辅因子结合位点来改变底物偏好以及增强HSDH的催化活性和热稳定性提供了有价值的见解。
