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

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.