Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments
Levin, Benjamin Joseph
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CitationLevin, Benjamin Joseph. 2019. Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe human gastrointestinal tract is home to trillions of microorganisms, and these microbes have important impacts on human health. Despite the many known links between gut microbiota composition and host biology, the molecular mechanisms underlying these interactions are largely unknown and there are no general strategies to elucidate them. Glycyl radical enzymes (GREs) had been previously identified in the human gut microbiota, but their functions in this environment are largely unknown. This thesis presents our work towards characterizing the chemistry performed by GREs and their impacts on human health. Our results illustrate how integrating a biochemical and mechanistic understanding of enzymes and metagenomic sequencing data can lead to insight into host-gut microbial symbioses.
Chapter 2 details the development and application of a metagenomic analysis workflow called chemically guided functional profiling to identify and quantify the GREs present in healthy human microbiomes. Our methodology combined biochemical knowledge of this protein superfamily with metagenomic sequencing data, and with this approach we computed abundances of specific GREs in these environments. We prioritized targets for biochemical characterization based on their abundance in the human gut and characterized two enzymes previously of unknown function, propanediol dehydratase (PD) and trans-4-hydroxy-L-proline dehydratase, demonstrating how chemically guided functional profiling can reveal novel biochemistry in complex microbial communities.
Chapter 3 describes the mechanistic characterization of PD. Intriguingly, a B12-dependent propanediol dehydratase (B12-PD) catalyzes the same overall reaction as PD but requires a different cofactor. To probe the mechanisms of these enzymes, we synthesized all four possible 18O-labeled 1,2-propanediol stereoisomers in high enantiomeric excess and used these compounds as substrates for PD and B12-PD. The results suggest PD catalyzes direct elimination of the C2 hydroxyl group from an initial substrate-based radical, while B12-PD instead mediates a 1,2-hydroxyl group migration. These experiments clarify how PD and other GREs important to human health perform challenging transformations and reveal mechanistic differences between GREs and B12-dependent enzymes that have evolved identical functions.
Chapter 4 details our efforts to further study protein families in metagenomic datasets. Complementing our work quantifying GREs in metagenomic reads, we searched for additional GREs in metagenomic assemblies and identified genes encoding for GREs not previously found in any sequenced microbial genome. We also applied chemically guided functional profiling to additional protein families. Our results highlight how existing metagenomics datasets can be mined to find both previously sequenced as well as novel enzymes.
Chapter 5 presents our discovery and initial characterization of a new GRE catalyzing the decarboxylation of indole-3-acetate to skatole (3-methylindole). The production of skatole by boar and cattle gut microbes is important to the agricultural industry, but the enzymes involved in this pathway have not been characterized. Using comparative genomics and knowledge of GRE biochemistry, we discovered two GRE indole-3-acetate decarboxylases (IADs) and validated that they catalyze the expected reactions. These findings are important for developing methods to inhibit skatole biosynthesis and for further characterizing the scope of reactions performed by GREs.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41121264
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