BilR is a gut microbial enzyme that reduces bilirubin to urobilinogen.

Metabolism of haem by-products such as bilirubin by humans and their gut microbiota is essential to human health, as excess serum bilirubin can cause jaundice and even neurological damage. The bacterial enzymes that reduce bilirubin to urobilinogen, a key step in this pathway, have remained unidentified. Here we used biochemical analyses and comparative genomics to identify BilR as a gut-microbiota-derived bilirubin reductase that reduces bilirubin to urobilinogen. We delineated the BilR sequences from similar reductases through the identification of key residues critical for bilirubin reduction and found that BilR is predominantly encoded by Firmicutes species. Analysis of human gut metagenomes revealed that BilR is nearly ubiquitous in healthy adults, but prevalence is decreased in neonates and individuals with inflammatory bowel disease. This discovery sheds light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining bilirubin homeostasis.

Discovery of the gut microbial enzyme responsible for bilirubin reduction to urobilinogen.

The degradation of heme and the interplay of its catabolic derivative, bilirubin, between humans and their gut microbiota is an essential facet of human health. However, the hypothesized bacterial enzyme that reduces bilirubin to urobilinogen, a key step that produces the excretable waste products of this pathway, has remained unidentified. In this study, we used a combination of biochemical analyses and comparative genomics to identify a novel enzyme, BilR, that can reduce bilirubin to urobilinogen. We delineated the BilR sequences from other members of the Old Yellow Enzyme family through the identification of key residues in the active site that are critical for bilirubin reduction and found that BilR is predominantly encoded by Firmicutes in the gut microbiome. Our analysis of human gut metagenomes showed that BilR is a common feature of a healthy adult human microbiome but has a decreased prevalence in neonates and IBD patients. This discovery sheds new light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining bilirubin homeostasis.

Gut Microbiome-Wide Search for Bacterial Azoreductases Reveals Potentially Uncharacterized Azoreductases Encoded in the Human Gut Microbiome.

The human gut is home to trillions of microorganisms that are responsible for the modification of many orally administered drugs, leading to a wide range of therapeutic outcomes. Prodrugs bearing an azo bond are designed to treat inflammatory bowel disease and colorectal cancer via microbial azo reduction, allowing for topical application of therapeutic moieties to the diseased tissue in the intestines. Despite the inextricable link between microbial azo reduction and the efficacy of azo prodrugs, the prevalence, abundance, and distribution of azoreductases have not been systematically examined across the gut microbiome. Here, we curated and clustered amino acid sequences of experimentally confirmed bacterial azoreductases and conducted a hidden Markov model-driven homolog search for these enzymes across 4644 genome sequences present in the representative Unified Human Gastrointestinal Genomes collection. We identified 1958 putative azo-reducing species, corroborating previous findings that azo reduction appears to be a ubiquitous function of the gut microbiome. However, through a systematic comparison of predicted and confirmed azo-reducing strains, we hypothesize the presence of uncharacterized azoreductases in 25 prominent strains of the human gut microbiome. Finally, we confirmed the azo reduction of Acid Orange 7 by multiple strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme Together, these results suggest the presence and activity of many uncharacterized azoreductases in the human gut microbiome and motivate future studies aimed at characterizing azoreductase genes in prominent members of the human gut microbiome. SIGNIFICANCE STATEMENT: This work systematically examined the prevalence, abundance, and distribution of azoreductases across the healthy and inflammatory bowel disease human gut microbiome, revealing potentially uncharacterized azoreductase genes. It also confirmed the reduction of Acid Orange 7 by strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme.