STRUCTURE AND FUNCTION OF DIVERSE GUT MICROBIAL ENZYMES

The human gut microbiome contains thousands of bacteria which conduct diverse chemical reactions by encoding millions of genes, many of which are absent from the human genome. These unique gut microbial proteins act on foods, therapeutics, and endogenous compounds, and their products are intricately related to human health, influencing disease states such as inflammatory bowel disease, cardiovascular disease, colorectal cancer, and depression. To understand the full impact of the gut microbiome on human homeostasis, unique bacterial gene products must be examined not only through microbiome sequencing efforts such as 16S rRNA sequencing and shotgun metagenomic sequencing, but also through biochemical efforts examining the enzymes themselves. Here, we first describe a structural metagenomics pipeline that utilizes metagenomics, in vitro structural biology, kinetics and inhibition, and chemical probe-based techniques to examine gut microbial enzymes and develop tools for manipulation of the gut microbiome in vivo. We then apply the pipeline to two classes of gut microbial enzymes: beta-glucuronidases (GUS) and bile salt hydrolases (BSH). First, we examine the effects of GUS enzymes on triclosan, a common antimicrobial compound present in consumer products such as soaps and toothpastes. Through a series of in vitro and ex vivo studies, we discover that a specific structural class of GUS enzymes reactivates triclosan from its inactive metabolite triclosan-glucuronide, causing gastrointestinal toxicity, and that a targeted GUS inhibitor alleviates this triclosan-induced colitis in mice. Next, we determine the structural underpinnings of BSH substrate specificity through crystal structures and mutagenesis studies, and we show that specific BSH activities can be leveraged to mediate C. difficile growth and infection. Additionally, we use BSH structures to develop an activity-based probe for proteomic analysis of BSHs in human fecal samples. Finally, we structurally characterize the heme oxygenase FlcD, a member of the flc pathway in Pseudomonas aeruginosa whose product is a copper-containing antibiotic. These structures reveal a unique iron binding mode and a mobile α-helix that facilitates substrate binding. Together, the studies conducted in this dissertation demonstrate the structural and functional diversity of microbial enzymes in the human gut and further establish the critical role these enzymes play in host physiology.