Metabolism of bilirubin to urobilinogen by human gut bacteria occurs by a branched, multi-enzyme pathway

Abstract

Bilirubin, the predominant product of heme catabolism in mammals, enters the intestine via the hepatobiliary system and subsequently is metabolized by the gut microbiome into multiple downstream derivatives, such as urobilinogen and stercobilinogen. Variation in levels of bilirubin and its derivatives are associated with inflammatory and metabolic disorders, but the microbial species and enzymes that metabolize bilirubin have remained largely unknown, which has precluded causal and mechanistic studies. Here, we demonstrate that metabolism of bilirubin to urobilinogen requires multiple distinct reactions carried out by separate enzymes. We find that bilirubin reductase (BilR), an enzyme that was recently discovered and proposed to convert bilirubin to urobilinogen, is specific for reducing the methine bridges of bilinoids, converting bilirubin to the previously uncharacterized intermediate divinylurobilinogen and mesobilirubin to urobilinogen. We identify bilinoid vinyl reductase (BilV), a flavin-dependent oxidoreductase of the Old Yellow Enzyme (OYE) superfamily, as the enzyme responsible for reducing the vinyl groups of bilirubin and divinylurobilinogen. Both BilR and BilV are capable of acting on multiple bilinoids, such that the reductions required to convert bilirubin to urobilinogen can proceed in multiple orders through a branched network of intermediates. BilV is widespread in human gut metagenomes and is broadly distributed across gut bacteria in a manner that overlaps with but does not mirror the distribution of BilR. These findings establish the identities of novel factors in bacterial bilirubin metabolism, delineate the branched architecture of the pathway, and enable defined studies to interrogate how such branched metabolism contributes to human health and disease.