The presence and induction of regioselective dehydroxylases dictate urolithin metabolism by Enterocloster species

Abstract Urolithins are a class of bioactive metabolites derived from the metabolism of dietary ellagitannins by the human gut microbiota. In the gut, urolithins are dehydroxylated regioselectively based on microbiota composition and activity. A single 9-hydroxy urolithin dehydroxylase (ucd) operon in gut resident Enterocloster species has been described to date; however, most enzymes in the urolithin metabolic pathway remain uncharacterized. Here, we investigate urolithin cross-feeding between members of the gut microbiota and discover a novel urolithin dehydroxylase in a subset of Enterocloster species. We show that urolithin intermediates, released by gut resident Gordonibacter species during ellagic acid metabolism, are dehydroxylated at both the 9- and 10-positions by E. asparagiformis, E. citroniae, and E. pacaense, but not E. bolteae. Using untargeted proteomics, we uncover a 10-hydroxy urolithin dehydroxylase operon, termed uxd, responsible for these species-specific differences in urolithin metabolism. By inducing uxd expression with diverse urolithins, we show that 9-hydroxy urolithins are required for uxd transcription and 10-position dehydroxylation. Collectively, this study reveals some of the genes, proteins, and substrate features underlying differences in urolithin metabolism by the human gut microbiota.