Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome due to their antimicrobial properties. Recently, a novel mechanism of BA metabolism by the gut microbiota was identified involving conjugation with various amino acids. Here, we show this unique biochemistry is mediated by bile salt hydrolase acting as an aminoacyltransferase or transpeptidase (BSH/T). Clostridium perfringens BSH/T rapidly performed acyl-transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at approximately pH 5.3. Amino acids selected for incorporation by CpBSH/T were pH-dependent and broad in range, though proline and aspartic acid conjugates were not observed. BA conjugation was widespread among gut bacteria but showed unique amino acid preferences depending on the organism. Members of the family Lachnospiraceae had closely related BSH/T amino acid sequences and exhibited similar amino acid conjugation profiles, while horizontal gene transfer of bsh in some species increased conjugation diversity. Tertiary structure mapping of BSH/T from different gut bacteria implicates active site structure in amino acid selectivity. The products of BSH/T aminoacyltransferase activity were potent antimicrobials but this depended on the amino acid conjugated, with phenylalanine and leucine-conjugates acting as the best inhibitors of other gut commensals and known pathogens. This inhibitory property translated in vivo, as oral gavage of mice with different microbially conjugated bile acids altered the cecal and fecal microbiomes. This study shows that transpeptidase activity of BSH/T is a widespread and common property of the enzyme that greatly diversifies the bile acid pool, with the potential to shape the human microbiome.

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