Discovery and Characterization of a Novel Reaction and Intermediate in Bilirubin Metabolism by Human Gut Bacteria

Abstract

An important pathway by which gut microbes affect host physiology is through the transformation of host-derived metabolites, which can both alter the levels of endogenous signaling agents and produce new bioactive molecules. One example is the bacterial transformation of bilirubin, the end product of mammalian heme catabolism, which is metabolized by gut microbes into a panel of derivatives including urobilinogen. Correlative studies have connected bilirubin and microbial bilirubin metabolites to increased risk for inflammatory and metabolic disorders, including inflammatory bowel disease. While we have known for decades that gut bacteria metabolize bilirubin, key aspects of this metabolism remain uncharacterized, including the bacteria and enzymes responsible and its influence on human biology. Here, we describe the discovery of a novel reaction and intermediate in bacterial bilirubin metabolism and the identification of the responsible enzyme. Previously, only a single bacterial enzyme for bilirubin metabolism had been characterized: bilirubin reductase (BilR), which was proposed to convert bilirubin to urobilinogen. Via an LC-MS based screen of a collection of human gut microbes, we discovered that BilR does not convert bilirubin to urobilinogen, rather it produces a novel compound, which we identify to be divinylurobilinogen. A second screen identified gut bacteria that further convert divinylurobilinogen to urobilinogen, completing the pathway. Finally, we used transcriptomics of divinylurobilinogen-exposed bacteria to identify the corresponding enzyme, which we name divinylurobilinogen reductase, and confirmed its activity using a gain-of-function approach. We find that this enzyme is prominent in all healthy human gut microbiomes, distributed across Bacillota and Actinomycetota phyla, and includes species not previously known to metabolize bilirubin. Together, our findings have established an updated model of bilirubin reduction to urobilinogen. Our work provides novel insight into a fundamental aspect of gut bacterial metabolism and enables mechanistic studies to define how microbial bilirubin metabolism influences host physiology and contributes to disease.