An official website of the United States government
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.
more »
« less
Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes
Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug’s direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmAandmfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103M−1s−1and KMfor metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of themfmABgenes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.
more »
« less
- Award ID(s):
- 2203750
- PAR ID:
- 10511596
- Editor(s):
- Rosenzweig, Amy
- Publisher / Repository:
- Proceedings of the National Academy of Sciences Press
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Edition / Version:
- 1
- Volume:
- 121
- Issue:
- 10
- ISSN:
- 0027-8424
- Subject(s) / Keyword(s):
- Metformin hydrolase nickel X-ray structure Pseudomonas kinetics mechanism
- Format(s):
- Medium: X Size: 2.4MB Other: html; pdf
- Size(s):
- 2.4MB
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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.more » « less
-
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.more » « less
-
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.more » « less
-
Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silicoAbstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2312652121
