Browsing by Subject "enzyme"

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    Elucidating the biodegradation pathway for the pharmaceutical metformin in wastewater: Implications in human and wastewater microbiomes
    (2023-12) Tassoulas, Lambros
    Metformin (1,1-dimethylbiguanide) is a wonder drug and a pervasive pollutant which is taken by type-II diabetes patients and more recently for improving obesity and cancer treatment outcomes. The drug’s direct mode of action is currently unknown but is thought to be dependent on microbial reactions to the drug. Large therapeutic doses (1-2 grams daily) and the global use of metformin result in over 100 million kilograms of the drug entering aquatic ecosystems each year. The biodegradation of metformin was known in wastewater treatment plants to be transformed by microbes to guanylurea but, prior to this work, no enzymes involved in the biodegradation had been identified. As a result of this work, the metformin biodegradation pathway has been completely elucidated starting with metformin hydrolysis to form dimethylamine and guanylurea by metformin hydrolase (MfmAB). The guanylurea hydrolase (GuuH) was discovered, which degrades guanylurea into guanidine, ammonia and carbon dioxide. Lastly, the enzyme CgdAB was discovered that acts as a carboxyguanidine deiminase bacteria use to assimilate nitrogen from guanidine. A secondary pathway of metformin biodegradation was also identified by discovering a biguanidase (BguH) that degrades biguanide and 1-methylbiguanide to form guanylurea, suggesting that metformin can also be demethylated by microbes. These enzymes were characterized by kinetics, X-ray crystallography or computational modelling, and bioinformatics. While the metabolism of metformin is now known in wastewater ecosystems, metabolism in the human gut has not been established but may contribute to the potency or therapeutic effect of the drug. Testing enzymes that are found in human gut microbes, homologous to the metformin hydrolase, did not show activity on metformin. However, a subset of these gut enzymes, from Gammaproteobacteria that hydrolyze agmatine, was potently inhibited by metformin and the natural product galegine, from which the drug was derived from. Agmatine is a known effector of human host metabolism and has been reported to augment metformin’s therapeutic effects for type-II diabetes. This gut-derived inhibition mechanism gives new insights on metformin’s action in the gut and may lead to significant discoveries in improving metformin therapy.
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    Isolating and Assaying Unspecific Peroxygenase and Flavin Binding Enzymes for in vitro Terpenoid Biosynthesis
    (2018-05) Hanson, Benjamin
    Terpenoids are an exceptionally large family of natural products, and contain numerous bioactive members that are pharmaceutically important. While most research into terpenoids and their metabolism has thus far occurred in non-fungal organisms, chiefly plants, Basidiomycota (mushroom forming fungi) are well known as prolific producers of bioactive sesquiterpenoids, such as the potent anticancer compounds Illudin M and S. While natural products have traditional been a huge driver of pharmaceutical discovery, this natural abundance is often hampered by very low expression in the native host and slow growth or rarity of the host itself. This drives up financial and environmental costs, and in many cases makes the production of otherwise useful natural products impracticable. One potential solution to this quandary is heterologous production of secondary metabolites in non-native hosts such as Escherichia coli and Saccharomyces cerevisiae. Another avenue is in vitro biocatalysis, wherein the biosynthetic enzymes of the native host are heterologously expressed, isolated, and used to perform synthesis outside of the cell. This approach avoids the fragility of the in vivo system and would allow the creation of combinatorial enzymatic pathways to create novel bioactive structures. With regards to constructing a sesquiterpenoid biosynthetic pathway, many stable terpene synthases have been isolated and shown to be active in vitro. However, the most well studied terpene scaffold modifying enzymes, cytochrome P450s, are notoriously difficult to heterologously express in active form. In order to obtain scaffold modifying oxygenases capable of being part of an in vitro terpenoid biosynthetic pathway, fungal oxygenases aside from cytochrome P450s were investigated. Specifically, unspecific peroxygenase from Agrocybe aegerita and flavin binding oxidoreductases from Δ6-protoilludene biosynthetic gene clusters were expressed and assayed against the sesquiterpene scaffold.