Mycothiol Conjugation Drives Biotransformation and Biodefluorination of Fluorotelomer Carboxylic Acids by Actinomycetota and Sludge Microbiomes

Microbial breakdown of poly- and perfluoroalkyl substances (PFASs) remains poorly understood, often hindered by mass balance discrepancies and unexplained fluoride release. Here we identify a previously unrecognized PFAS biodefluorination mechanism mediated by mycothiol (MSH), a ubiquitous thiol antioxidant produced by Actinomycetota . Using high-resolution mass spectrometry and non-target analysis, MSH-mediated conjugation is unraveled as a dominant biotransformation pathway for two representative fluorotelomer carboxylic acids (6:2 and 5:3 FTCAs) in Rhodococcus jostii RHA1 and other MSH-producing species. Conjugation proceeds through nucleophilic substitution and β-elimination in 6:2 FTCA, releasing two fluoride ions, and through Michael addition in the unsaturated analogue of 5:3 FTCA. These reactions are catalyzed by the conserved enzyme mycothiol S-transferase (MST), which contains a conserved His47–Asp150–His154 tripod motif essential to activate MSH for nucleophilic attack. Subsequent hydrolysis by mycothiol S-conjugate amidase (Mca) yields mercapturic acid derivatives, with more than 20 novel downstream thiol metabolites detected. Time-series experiments confirmed that MSH conjugation accounted for up to half of FTCA removal and fluoride release, resolving major fluorine mass balance gaps. The conjugation process also interlinks with chain-shortening “one-carbon removal” routes, forming a cascading metabolic network. Importantly, conjugated metabolites were consistently detected in activated sludge microbiomes, and metagenomic surveys revealed more than 100,000 MST homologs worldwide. Collectively, these findings establish MSH-mediated conjugation as a widespread and mechanistically distinct route of PFAS biodefluorination, highlighting new opportunities for microbial PFAS removal in natural and engineered systems.