The dietary ellagitannin metabolite urolithin A is produced by a molybdenum-dependent dehydroxylase encoded by prevalent human gut <i>Enterocloster</i> spp

Abstract Urolithin A (uroA) is a polyphenol derived from the multi-step metabolism of dietary ellagitannins by the human gut microbiota that can affect host health by stimulating mitophagy. Most individuals harbor a microbiota capable of uroA production; however, the mechanisms underlying the dehydroxylation of its catechol-containing dietary precursor (uroC) are unknown. Here, we use a combination of untargeted bacterial transcriptomics, proteomics, and comparative genomics to uncover an inducible uroC dehydroxylase ( ucd ) operon in Enterocloster spp. We show that Enterocloster spp. are sensitive to iron chelation by uroC, and dehydroxylation to uroA rescues growth by disrupting the iron-binding catechol. Importantly, only microbiota samples actively transcribing ucd could produce uroA, establishing ucd -containing Enterocloster spp. as keystone urolithin metabolizers. Overall, this work identifies Enterocloster spp. and the ucd operon as main contributors to uroA production and establishes a multi-omics framework to further our mechanistic understanding of polyphenol metabolism by the human gut microbiota.