Discovery of BilV reveals a multienzymatic basis for bilirubin reduction across vertebrate gut microbiomes

Abstract Gut bacteria reduce bilirubin to urobilinogen, allowing it to be excreted through feces and urine, but studies have long noted a heterogeneous mixture of partially reduced bilirubin-derived intermediates, suggesting that multiple enzymes are involved. Here we identify bilirubin vinyl reductase (BilV), a novel Old Yellow Enzyme family reductase encoded in the genomic neighborhood of the known bilirubin reductase ( bilR ). Using heterologous expression and LC-MS/MS, we show that BilR acts on the methine bridges in the bilirubin reduction pathway; co-expression with BilV enables vinyl-group reduction and complete conversion to urobilinogen. In bacterial genomes, bilV co-occurs primarily with the bilR -insertion subtype and is largely absent alongside bilR -short. Analysis of 1,197 gut metagenomes across 14 vertebrate species reveals that this differential co-occurrence shapes pathway availability across hosts: carnivores and omnivores carry balanced bilR and bilV , whereas avian microbiomes, dominated by bilR -short, are depleted for bilV . These findings establish that bilirubin reduction to urobilinogen involves two enzymes with complementary regioselectivity, and that their distribution across vertebrate gut microbiomes varies in concert with host bile pigment chemistry.