School of Pharmacy, Medical Biology Centre, Queen's University Belfast, BT9 7BL Northern Ireland, UK.
School of Biochemistry and Cell Biology, University College Cork, Cork T12 YT20, Ireland; APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland.
Structure. 2023 May 4;31(5):629-638.e5. doi: 10.1016/j.str.2023.02.014. Epub 2023 Mar 23.
Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.
胆盐水解酶（BSHs）目前被作为人类代谢调节剂的靶标酶和农场动物的生长促进剂进行研究。了解底物特异性的结构特征对于抑制剂的设计是必要的。在这里，我们使用了包括质谱、突变、分子动力学模拟、机器学习和晶体学在内的多学科工作流程，证明了在乳杆菌 BSH 中的底物特异性，乳杆菌 BSH 是人类和农场动物肠道中最丰富的酶。我们表明，带有牛磺酸头部和去羟基固醇环的底物更倾向于水解。一个回归模型，将各种酶突变体中各种底物的水解相对速率与残基-底物相互作用能相关联，指导了对底物结合和特异性的结构决定因素的鉴定。此外，我们还发现来自另一个 BSH 前体的 T208 调节水解。该设计的工作流程可用于快速、全面地表征具有广泛底物的酶。
