Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in hu-mans 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 spec-trometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demon-strate 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 charac-terization of enzymes with a broad range of substrates.

Automated summary

This study investigated the substrate specificity of bile salt hydrolases (BSHs), which are potential target enzymes for drugs and feed additives. The researchers found that Lactobacillus salivarius BSH showed preference for substrates with a taurine head and a dehydroxylated sterol ring. They used a regression model and crystallography to identify the structural determinants of substrate binding and specificity. Additionally, they discovered that a different BSH subunit, T208, also regulated the hydrolysis activity. This multidisciplinary workflow can be used to characterize enzymes with diverse substrates quickly and comprehensively.