Discovery and Characterization of Gut Microbial Enzymes Involved in Xenobiotic Metabolism
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Maini Rekdal, Vayu
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CitationMaini Rekdal, Vayu. 2020. Discovery and Characterization of Gut Microbial Enzymes Involved in Xenobiotic Metabolism. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe trillions of microorganisms inhabiting the human gastrointestinal tract (the gut microbiota) harbor metabolic capabilities that expand the range of chemistry taking place in the human body. Gut microbial metabolism directly modifies the chemical structures of diverse ingested and endogenous compounds, making the gut microbiota a key player in pharmacology, nutrition, and host biology. However, few gut microbial transformations have been connected to specific strains and enzymes, limiting our ability to harness microbial chemistry for human health. This thesis describes the discovery and characterization of enzymes that break down the drug L-dopa, catecholamine neurotransmitters, and dietary phytochemicals. This work has provided opportunities to manipulate and study gut microbial metabolism and has uncovered catalytic functions and metabolic pathways that may be broadly relevant to microbial life.
Chapter 2 describes the discovery of organisms and enzymes that metabolize Levodopa (L-dopa), the main treatment for Parkinson’s disease. This drug alleviates many of the symptoms associated with Parkinson’s disease but suffers from high interindividual variability in both efficacy and side effects. Although the gut microbiota has been implicated in L-dopa efficacy and was known to metabolize L-dopa, the microbial enzymes responsible were poorly understood. A major pathway for microbial L-dopa metabolism involves decarboxylation of L-dopa into dopamine followed by dehydroxylation into m-tyramine. We used genome mining, genetics, and biochemistry to identify and characterize a promiscuous tyrosine decarboxylase (TyrDC) from the human gut microbe Enterococcus faecalis that directly decarboxylates L-dopa into dopamine. This gene predicted L-dopa metabolism by microbial strains and by complex gut microbiota samples from Parkinson’s patients and neurologically healthy individuals. We next evaluated the activity of known host decarboxylase inhibitors in microbial L-dopa decarboxylation. In contrast to its potency towards host metabolism, the commonly prescribed decarboxylase inhibitor carbidopa only weakly microbial L-dopa decarboxylation, suggesting that some metabolism may be unaccounted for in the current treatment regimen. This observation led us to identify (S)-α-fluoromethyltyrosine, a small molecule that selectively inhibits microbial L-dopa decarboxylation by isolated strains and microbial communities. Finally, we uncover that the gut bacterium Clostridium sporogenes uses enzymes involved in aromatic amino acid metabolism to degrade L-dopa via an alternative pathway.
Chapter 3 describes our work identifying and characterizing microbial strains, genes, and enzymes involved in dehydroxylating the catechol ring of dopamine, a human neurotransmitter and the intermediate in the two-step microbial metabolism of L-dopa into m-tyramine. We used enrichment culturing from complex human fecal samples to isolate a strain of Eggerthella lenta that dehydroxylates dopamine to give m-tyramine. We then used RNA-sequencing, comparative genomics, chemical genetics, and in vitro biochemistry to identify a molybdenum-dependent enzyme (named the dopamine dehydroxylase, or Dadh) involved in this reaction. Bioinformatics analysis revealed that a single nucleotide polymorphism in the gene encoding this enzyme correlated with metabolism by isolated strains and complex microbial communities.
Chapter 4 expands on our discovery of Dadh. Dopamine dehydroxylation is just one of many examples of catechol dehydroxylation, a poorly understood but prominent transformation in gut microbial metabolism of endogenous compounds, dietary small molecules, and pharmaceutical drugs. We first characterized the substrate scope and regulation of Dadh and found a remarkable specificity for the catecholamine scaffold. We also demonstrated that dopamine can serve as an alternative electron acceptor for E. lenta, providing a potential physiological explanation for this high specificity. To identify strains capable of metabolizing catechols beyond dopamine, we screened a collection of gut bacteria for metabolism of catecholic drugs, dietary compounds, and siderophores. This screen unveiled that Gordonibacter pamelaeae 3C dehydroxylates DOPAC and that the closely related E. lenta dehydroxylated diet-derived hydrocaffeic acid and (+)-catechin. RNA-sequencing and comparative genomics revealed candidate molybdenum-dependent enzymes involved in these reactions, and the native purification of the hydrocaffeic acid dehydroxylase (Hcdh) strongly supported a model in which each catechol is metabolized its own dedicated gut microbial enzyme. Phylogenetic and sequence similarity network analyses established catechol dehydroxylases as a new class of molybdenum-dependent enzymes harboring vast uncharacterized diversity among gut microbes and environmental microbes alike. Finally, we found that catechol dehydroxylation was present among gut microbiotas of mammals representing diverse diets and phylogenetic origins, reinforcing that this activity can take place in habitats beyond the human gut. This suggested that the chemical strategies used to enable microbial survival and interactions in the human gut are relevant to a broad range of species and habitats.
Chapter 5 describes our progress towards mechanistic and biochemical characterization of catechol dehydroxylases. Based on the Dadh and Hcdh substrate scope and phylogenetic analysis in Chapter 4, we proposed a mechanism for catechol dehydroxylation that represented a new strategy for aromatic dehydroxylation. Deuterium incorporation experiments investigating hydrocaffeic acid dehydroxylation in cell lysates supported our mechanistic proposal, revealing that a single deuterium was incorporated at high levels (>95%) into the aromatic ring of the dehydroxylated product. To enable further mechanistic and biochemical characterization, we turned to heterologous expression. We cloned and expressed the gut microbial dopamine dehydroxylase (Dadh), hydrocaffeic acid dehydroxylase (Hcdh), catechin dehydroxylase (Cadh), DOPAC dehydroxylase (Dodh), catechol lignan dehydroxylase (Cldh), and a putative dehydroxylase from the soil microbe T. aromatica (Tardh). Our efforts involved extensive testing of different plasmids across different heterologous and media conditions. However, no constructs yielded soluble, active protein, suggesting further work is necessary to overcome the challenges associated with the complex maturation and assembly of molybdenum-dependent enzymes.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365731
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