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

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.