Identification and characterization of unique enzymatic transformations by gut microbes

Abstract

The human gut microbiome is a diverse community of microorganisms that catalyze millions of unique biotransformations of both endogenous and exogenous compounds. The production and modification of these microbial products can influence both other gut microbes and the human host, having impacts on health and disease. Characterizing these biotransformations is an important part of understanding the functions of the gut microbiome and a key step in the development of microbiome-directed therapies. In this thesis, I describe my work identifying and characterizing several unique microbial enzymes and the conditions necessary for their activity. In chapter 2, I describe our identification of a steroid 21-dehydroxylase present in gut microbial species Eggerthella and Gordonibacter that transforms glucocorticoid steroids into progestins. My work in this chapter focuses on understanding the role of hydrogen production by E. coli and other bacteria in facilitating this reduction, characterizing the genetic and metabolic regulation of the steroid 21-dehydroxylase, and developing protein purification parameters both to confirm our identified enzyme and to provide a framework for subsequent mechanistic studies. In chapter 3, we introduce a new biochemical transformation for microbiome— sulfonation. Sulfonation is an important biochemical transformation that has long been attributed solely to host metabolism. We showed that the gut commensal Bacteroides thetaiotaomicron encodes for a sulfotransferase (SULT) enzyme capable of catalyzing the sulfonation of cholesterol and structurally related steroidal molecules (BT0416 or BtSULT). Our discovery and characterization of this enzyme revealed that it is structurally selective for substrates with a “planar” 5α-trans or 5-ene A/B ring structure within the steroidal core. Additionally, accompanying in vivo studies in monocolonized germ free (GF) mice showed that the presence of this bacterial gene increased cholesterol sulfate (ChS) levels and reduced T cell migration to the mesenteric lymph nodes, showing the potential for sulfated products of the gut microbiome to influence host biology. In chapter 4, we follow up on our discovery of BtSULT, seeking to identify and study new sulfotransferases across the microbiome. Sulfated metabolites have important roles in regulation of metabolism, immune cell function and migration, and disease phenotypes; therefore, understanding the role of the microbiome in producing sulfated metabolites is an important area of study. To identify and study SULTs across the microbiome, we leveraged PAPS biosynthesis to integrate a stable isotope labeled sulfate moiety into sulfated metabolites both in bacterial monoculture and in vivo mouse samples. Using a combination of comparative methods and high-resolution mass spectrometry, we identified hundreds of sulfated features in mouse tissues and several new chemical classes from bacterial cultures. We identified sulfated hydroxy fatty acids as a new type of microbiome-derived sulfated metabolite and identified a candidate sulfotransferase enzyme encoded in the genome of Eubacterium ramulus ATCC 29099. The data from these exploratory studies will inform future investigations of other classes of sulfated metabolites and provide a framework for studying their SULTs. In all, these studies contribute to our understanding of gut bacterial enzymes, their metabolic and genetic regulation, and the biological activities of their products. The tools used in these studies will facilitate future studies of gut bacterial enzymes and the small molecule metabolites that they produce.