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Abstract
The human gut microbiome is one of the most biochemically rich ecosystems in nature, housing approximately 1000 bacterial species, tens of trillions of cells, and millions of genes. Gut microbes are intimately associated with health outcomes that range from diabetes to depression, yet we have only begun to understand the chemical and biological mechanisms the gut microbiome utilizes to impact host health. One space of this biochemical dark matter is gut bacterial β-glucuronidases (GUSs), glycoside hydrolases that metabolize a myriad of glucuronides in the human gut associated with the dose-limiting toxicities of essential therapeutics. Here we show our efforts to characterize and inhibit gut bacterial GUSs. Structure- and function-guided analysis of GUS genes from the Human Microbiome Project Stool Sample Database revealed three GUSs in a single gut microbe, enabled the discovery of a family of GH2 β-galacturonidases (GalAses) and hybrid GH2 GUS/GalAses, and unearthed a family of novel FMN-binding GUSs in the human gut. Structurally, gut bacterial GUSs demonstrate remarkable diversity for a single enzyme family, in which tertiary structure is conserved, but quaternary structure is highly diverse and a key predictor of substrate specificity. Lastly, we determined the mechanism and performed a structure-activity-relationship of piperazine-containing GUS inhibitors. Inhibition by piperazine-containing inhibitors proceeds via a unique substrate-dependent mechanism that appears to trap GUS during catalysis. We further show that piperazine-containing approved drugs act via the same mechanism, suggesting that approved drugs have off targets in the gut microbiome. Taken together, the work outlined in this dissertation advances our understanding of the structure, function, and inhibition of gut bacterial GUSs and raises many questions about the core function of bacterial GUS in host physiology.
