Discovery and Characterization of a Prominent Gut Microbial Glycyl Radical Enzyme Responsible for 4-Hydroxyproline Metabolism

Abstract

The human gut is one of the most densely populated microbial habitat on Earth and the gut microbiota is extremely important in maintaining health and disease states. Advances in sequencing technologies have enabled us to gain a better understanding of microbiome compositions, but the majority of microbial genes are not functionally annotated. Therefore, the molecular basis by which gut microbes influence human health remains largely unknown. Mechanistic studies connecting sequences to functions remain a priority in this field. In this thesis, we describe the discovery and characterization of a new glycyl radical enzyme (GRE) responsible for anaerobic metabolism of an abundant host-derived amino acid. From our work on this enzyme, we uncovered a widely distributed metabolic capability for trans-4-hydroxy-L-proline (Hyp) dehydration in the gut microbiome. The activity of this novel GRE was proposed based on its genomic context and high sequence similarities to characterized GRE eliminases. This GRE was predicted to be a Hyp dehydratase (HypD) that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylate (P5C). Through in vitro reconstitution of the GRE and its activating enzyme, we experimentally validated its proposed function. Hyp metabolism was demonstrated specifically in HypD-encoding Clostridiales in culture-based experiments. HypD was found among common gut isolates and was detected in all stool metagenomes analyzed. Overall, the work covered in this Chapter has expanded known reactivities in the GRE superfamily and revealed the enzyme responsible for anaerobic Hyp metabolism. Chapter 3 details work from a close collaboration with the Drennan lab and help from the Raines lab at MIT. Structural elucidation of substrate-bound HypD was achieved through X-ray crystallography by Lindsey Backman (Drennan lab) and Hyp conformation was calculated by Dr. Brian Gold (Raines lab). The HypD crystal structure informed site-directed mutagenesis of conserved residues. Biochemical characterization of these protein mutants revealed the importance of these residues in activity. The structural and biochemical work provided insight into the molecular basis of Hyp dehydration. The unique features of this elimination reaction were highlighted and a mechanism was proposed for HypD. Chapter 4 describes the phylogenetic analyses of HypD sequences along with analyses of their genome neighborhoods. Hyp-degrading bacterial communities from environmental samples were obtained through enrichment culturing. Determination of community compositions revealed species closely related to sequenced HypD-encoding isolates. Detection of hypD in these cultures provided support for HypD to be the principle enzyme responsible for anaerobic Hyp metabolism. In Chapter 5, we present work toward identifying the physiological role of HypD among gut Clostridiales and Bacteroidales. We demonstrated that HypD is part of Stickland fermentation and upstream of L-proline reduction. With help from Prof. Aimee Shen (Tufts University), Clostridioides difficile deletion mutants were generated to show HypD is necessary for Hyp metabolism. From comparative transcriptomics of C. difficile, pathways were found to be differentially regulated by Hyp and Pro. The activity of HypD from Bacteroides vulgatus was verified in vitro so we attempted to detect Hyp metabolism in gut Bacteroidales. Bacteroides genetics, growth experiments, and competitive colonization of mice part of a collaboration with Prof. Laurie Comstock (Harvard Medical School) are described. At the end of this Chapter, we discuss in depth potential roles of Hyp metabolism in microbial physiology and its impact on host biology.