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Metformin is one of the most regularly prescribed Type II diabetes drugs in the world, and its use is likely to expand as diabetes diagnoses rise globally. This drug and its main degradation byproduct, guanylurea, are not fully metabolized by humans and cannot be removed through conventional water treatment processes. These compounds have been detected in coastal waters around the world and are currently considered emerging pollutants. The goal of this research was to examine the catalytic mechanism and substrate specificity of Guanylurea Hydrolase (GuuH), a recently discovered enzyme that converts guanylurea to ammonia and guanidine. Bioinformatic analyses were conducted to predict the active site and three-dimensional structure of GuuH. Site-directed mutagenesis was performed to construct mutants in amino acids predicted to be part of the enzyme's catalytic triad and substrate binding site. The mutants created were K138R, N141K, E211D, E211Q, and E211N. The wild-type and mutant enzymes were purified using His-tag affinity chromatography. Enzyme activity was assessed by measuring ammonia released using Berthelot assays. The results showed that the K138R mutant had similar specific activity compared to the wild-type GuuH when reacting with guanylurea, while E211N and E221D showed low specific activity under the same conditions. All of the enzymes had no detectable activity when reacting with biuret, which suggests they have low affinity for this substrate. Future work will focus on kinetic analyses of the wild-type and K138R enzymes and additional mutagenesis to identify the amino acids that determine the substrate specificity to the enzyme. Understanding GuuH's catalytic activity and substrate specificity is essential to using this enzyme in the development of biotechnological applications for water treatment. 

Anthropogenic biguanide compounds are widely used in agriculture, industry, and medicine, making them prevalent in the environment. The extensive use of compounds like cyanoguanidine, guanylurea, and metformin has led to their accumulation as pollutants in waterways. This research focused on the microbial degradation of cyanoguanidine, a common biguanide compound used as an additive in agricultural products and frequently present as an impurity in the production of pharmaceuticals such as metformin. Due to its extensive use, cyanoguanidine is classified as a persistent and mobile pollutant commonly detected in wastewater. The presence of cyanoguanidine in wastewater is hypothesized to exert selective pressure on microbial communities, driving the evolution of bacteria capable of metabolizing this compound as their sole nitrogen source. We hypothesized that sludge-derived microbial cultures could serve as a reservoir for isolating cyanoguanidine-degrading bacteria. To test this, enrichment cultures were established using thick and return-activated sludge from a municipal wastewater treatment plant. Microbial media containing cyanoguanidine as the sole nitrogen source was used to support bacterial growth. Aliquots of enrichment cultures were plated on minimal media containing cyanoguanidine to isolate bacteria capable of its metabolism. HPLC analysis was employed to monitor and quantify cyanoguanidine degradation, revealing its conversion to guanylurea, CO2, and NH3 by two bacterial isolates. Genome sequencing identified these cyanoguanidine degraders as Pseudomonas stutzeri and Pseudomonas mendocina. Bioinformatic analyses identified candidate genes involved in the degradation pathway, including nitrile hydratase and guanylurea hydrolase enzymes in both bacterial genomes. Although further investigation is needed to confirm the role of nitrile hydratase in cyanoguanidine metabolism, this study advances our understanding of microbial biguanide degradation. These findings contribute to the development of biotechnological strategies for removing biguanide pollutants from the environment. 

Biguanides are organonitrogen compounds prevalent in wastewater due to their widespread use in agricultural additives, pharmaceuticals, and personal care products. Metformin (1,2-N-dimethylbiguanide) is the most prescribed type II diabetes drug in the world and has been detected at significant levels in coastal waters and rivers worldwide. While recent studies reported the isolation of metformin-degrading bacteria and uncovered its catabolic pathway, the biodegradation of other biguanides and the connections between their metabolic pathways remain unexplored. This study describes establishing a collaborative undergraduate research program amongst three universities that focused on investigating the biodegradation of biguanides using microbes from soils and wastewater. As part of a course undergraduate research experience (CURE) and a summer research program, students prepared microbial enrichment cultures using soils from diverse locations and activated sludge samples from wastewater treatment facilities. The samples were grown on a minimal medium with biguanides including metformin, 1-N-methylbiguanide, and cyanoguanidine as the sole nitrogen sources for growth. Biodegradation and microbial growth were monitored by HPLC analyses, colorimetric assays, and optical density, respectively. Microbial consortia capable of degrading multiple biguanides were obtained and further characterized by genome sequencing and bioinformatics analyses. Bacterial strains that metabolized biguanides independently of microbial consortia were isolated and examined for the presence of candidate genes and enzymes potentially involved in biguanide metabolism. Guanylurea hydrolase, an enzyme commonly found in several biguanide catabolic pathways, was selected for further mutagenesis and biochemical analyses. The results of these studies suggest that the microbial catabolism of biguanide compounds shares common guanylurea and guanidine intermediates as well as the enzymes involved in their metabolism. The CURE and summer research activities were implemented over two years in introductory as well as intermediate courses at the participating universities. Preliminary assessment results show students' learning gains in collaboration skills, data interpretation, and the application of microbial enzymes to enhance wastewater treatment processes. 

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonas mendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today.