Enzymatic Basis for the Oxidative Branch of Aromatic Amino Acid Fermentation Leading to <i>p</i> ‐cresol Formation

The phenolic metabolite p-cresol is a byproduct of tyrosine fermentation by certain strictly anaerobic bacteria, including the human gut pathogen Clostridium difficile, with toxic effects on the host and intestinal microbiota. The only enzyme in this biochemical pathway characterized to date is the glycyl radical enzyme p-hydroxyphenylacetate (HPA) decarboxylase (HPAD), which catalyzes the terminal step. Here we report the identification and characterization of enzymes for anaerobic degradation of tyrosine to HPA in the model p-cresol-producing bacterium Clostridium scatologenes. In this pathway, tyrosine is first converted to p-hydroxyphenylpyruvate (HPP), followed by net oxidation to HPA by a trio of enzymes HPP: ferredoxin oxidoreductase (HpdDEFG), phosphate HPA-transferase (HpdJ) and HPA kinase (HpdK). Each step is thermodynamically reversible, and the pathway is coupled to net generation of ATP and reduced ferredoxin. A bioinformatics search reveals that gene clusters containing a similar trio of enzymes are widespread in anaerobic Firmicutes bacteria, suggesting analogous pathways for the degradation of other amino acids. These findings clarify the oxidative pathways by which anaerobic gut bacteria convert aromatic amino acids into a major class of aromatic metabolites including HPA and p-cresol, deepening our understanding of microbiota-host metabolic interactions.