Chemical proteomics reveals regulation of bile salt hydrolases via oxidative post-translational modifications

The gut microbiome is the vast, diverse ecosystem of microorganisms that inhabits the human intestines and provides numerous essential functions for the host. One such key role is the metabolism of primary bile acids that are biosynthesized in the host liver into a plethora of secondary bile acids produced by gut bacteria. These metabolites serve as both antimicrobial and chemical signaling agents within the host. The critical microbial enzyme that plays a gatekeeping role in secondary bile acid metabolism is bile salt hydrolase (BSH), a cysteine hydrolase that is primarily known for its deconjugating and reconjugating activities on bile acid substrates. Despite the crucial nature of these biotransformations, regulation of BSH activity is not well understood. Here, we found that the catalytic cysteine 2 (Cys2) within the BSH active site exists in multiple sulfur oxidation states including sulfenic acid (Cys-SOH). Importantly, we show this reversible oxidative post-translational modification (oxPTM) ablates BSH catalytic activity. We have leveraged this discovery to develop a chemoproteomic platform featuring a sulfenic acid-reactive bile acid probe to profile BSH Cys2 oxPTMs throughout the gut microbiome. Our results reveal that though most gut microbiota-associated BSHs exist in the active Cys2-SH state, some are preferentially and reversibly inactivated in the Cys2-SOH state. This reversible oxidation of Cys2 may serve as a general mechanism to regulate BSH activity in vivo in response to a changing physiological environment.