Browsing by Author "Tassoulas, Lambros"
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Item Elucidating the biodegradation pathway for the pharmaceutical metformin in wastewater: Implications in human and wastewater microbiomes(2023-12) Tassoulas, LambrosMetformin (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.Item Novel Discrimination Of Biuret And Triuret Degradation By Enzymatic Deamination, Regulation And Significance For Slow-Release Nitrogen Fertilizers(2019-08) Tassoulas, LambrosTriuret (carbonyl diurea) is an impurity of industrial urea fertilizer (<0.2%) which results in several hundred thousand tons applied, worldwide, each year on agricultural lands. Triuret has been described in the literature as early as 1870 when it was first synthesized and, although the natural source of triuret is unknown, it is hypothesized to be from oxidative uric acid metabolism. The biodegradation of triuret was known but, prior to this work, no enzymes had been identified. The triuret decomposing enzyme (TrtA) was discovered by observing two paralogs in gene clusters, one of which was well characterized as a biuret hydrolase (BiuH). TrtA, an amidohydrolase from the isochorismatase hydrolase like superfamily (IHL), similarly to BiuH, has a catalytic efficiency (kcat/Km) of 6x10^5 (M-1s-1) and a Km for triuret in the μM range with narrow substrate specificity. Crystal structures of TrtA in apo and holo form were solved which show an intriguing comparison with the BiuH structure where second shell residues around the near-identical active sites direct the specificity for each native substrate. With this discovery, the regulation of the triuret degradation operon, which can direct the mineralization of triuret completely to ammonium and carbon dioxide, was investigated in Herbaspirillum} sp. BH-1. The operon was shown to be specifically induced by biuret or triuret and suppressed in nitrogen sufficient conditions. Interestingly, the triuret hydrolase genes in the RefSeq database (<1%) come largely from plant-associated bacteria that can nodulate or are known endophytes and the broader context of this metabolism remains to be seen. As metabolism of triuret is relatively rare, and triuret is far less soluble in water than urea or ammonium nitrate, triuret may be considered a potential slow release N fertilizer.