Discovery and Characterization of Gut Bacterial Polyphenol Dehydroxylases

Abstract

Polyphenols found in plant-based foods are associated with various health benefits, including reduced inflammation and healthy aging. Notably, gut bacteria extensively metabolize polyphenols, altering their bioactivities and bioavailabilities. However, the bacterial enzymes responsible for polyphenol metabolism remain largely unknown. In my thesis work, we sought to advance our understanding of gut bacterial polyphenol metabolism by discovering and characterizing enzymes that dehydroxylate polyphenols, a transformation of significant chemical interest. Using an interdisciplinary approach that integrated bioinformatics, protein biochemistry, spectroscopy, structural characterization, and microbiology, we discovered molybdenum-dependent polyphenol dehydroxylases, including several catechol dehydroxylases and a xanthine dehydrogenase, that generate health-relevant gut metabolites. We further characterized catechol dehydroxylases by elucidating the active site environment, identifying key amino acid residues involved in catalysis and substrate recognition, and proposing a catalytic mechanism. Altogether, my thesis establishes a foundation for future investigations into polyphenol dehydroxylases and their impacts on human health. Chapter 2 describes our metatranscriptomics-guided discovery of a prominent catechol dehydroxylase from the human gut microbiome. We identified a highly expressed and prevalent uncharacterized catechol dehydroxylase (Gp Hcdh) in the human gut and determined that its substrate is hydrocaffeic acid (HCA), a common metabolite derived from polyphenols found in plant-based foods like coffee and cruciferous vegetables. Gp Hcdh activity was linked to anaerobic respiration in the encoding Gordonibacter strains and correlated with host inflammation in a human cohort. Together, these findings highlight the utility of metatranscriptomics-guided discovery and suggest a potential connection between polyphenol metabolism and host inflammation. In Chapter 3, we used Gp Hcdh as a model to investigate the molybdenum environment and catalytic mechanism of catechol dehydroxylase. X-ray absorption spectroscopy and mutagenesis identified that the Cys157 thiolate ligates the molybdenum ion. These insights informed computational modeling, which suggested a unique mechanism involving dearomatization and a 1,2-hydride shift. Additionally, these analyses highlighted a catalytic role for the active site Asp210 carboxylate as a general base, which was confirmed through mutagenesis. The proposed mechanism explains the requirement for a catechol motif in catalysis and warrants further experimental validation. In Chapter 4, we determined the first structure of a catechol dehydroxylase (Gp Hcdh) using cryo-electron microscopy (cryo-EM), revealing the active site environment and substrate-binding funnel. Guided by this structure, mutagenesis and docking identified ten key funnel residues, including Arg371 and Arg488, which likely engage the substrate via ionic interactions. Using these insights, we discovered a previously uncharacterized enzyme (Hpbh) that dehydroxylates 4-(3-hydroxypropyl)benzene-1,2-diol, a novel polyphenol metabolite. Expanding this approach, we identified and validated HCA dehydroxylases from diverse environmental bacteria, showing this activity extends beyond Coriobacteriia in the human gut. Lastly, Chapter 5 describes our efforts to identify gut bacterial enzymes involved in sequential dehydroxylation of ellagic acid (EA) to urolithin A, an anti-inflammatory metabolite and dietary supplement. Using differential gene expression analyses, we identified five enzymes from three gut bacterial genera: a lactone hydrolase (Eah) and three catechol dehydroxylases (Eadh1/2/3) from Gordonibacter and Ellagibacter, and a distinct molybdenum-dependent enzyme (Ucdh) within the xanthine dehydrogenase family from Enterocloster. Biochemical characterization revealed that substrate specificity of each dehydroxylase shapes the EA metabolic pathway. Notably, Ucdh, which does not require catechol, likely operates via a different catalytic mechanism from catechol dehydroxylase. Finally, genes encoding urolithin A-producing enzymes were depleted in the gut microbiomes of inflammatory bowel disease (IBD) patients, suggesting that gut bacterial metabolism of EA may differentially influence host inflammation in this context.