Filling in the Gaps in Metformin Biodegradation: a New Enzyme and a Metabolic Pathway for Guanylurea

Abstract

The widely prescribed pharmaceutical metformin and its main metabolite guanylurea are currently two of the most common contaminants in surface and wastewater. Guanylurea often accumulates and is poorly, if at all, biodegraded in wastewater treatment plants. This study describes Pseudomonas mendocina strain GU isolated from a municipal wastewater treatment plant using guanylurea as its sole nitrogen source. The genome was sequenced with 36-fold coverage and mined to identify guanylurea degradation genes. The gene encoding the enzyme initiating guanylurea metabolism was expressed, the enzyme purified and characterized. Guanylurea hydrolase, a newly described enzyme, was shown to transform guanylurea to one equivalent of ammonia and guanidine. Guanidine also supports growth as a sole nitrogen source. Cell yields from growth on limiting concentrations of guanylurea revealed that metabolism releases all four nitrogen atoms. Genes encoding complete metabolic transformation were identified bioinformatically, defining the pathway as follows: guanylurea to guanidine to carboxyguanidine to allophanate to ammonia and carbon dioxide. The first enzyme, guanylurea hydrolase, is a member of the isochorismatase-like hydrolase protein family that includes biuret hydrolase and triuret hydrolase. Although homologs, the three enzymes show distinct substrate specificities. Pairwise sequence comparisons and the use of sequence similarity networks allowed fine structure discrimination between the three homologous enzymes and provided insights into the evolutionary origins of guanylurea hydrolase. IMPORTANCE Metformin is a pharmaceutical most prescribed for type 2 diabetes and is now being examined for potential benefits to COVID-19 patients. People taking the drug pass it largely unchanged and it subsequently enters wastewater treatment plants. Metformin has been known to be metabolized to guanylurea. The levels of guanylurea often exceed that of metformin, leading to the former being considered a “dead end” metabolite. Metformin and guanylurea are water pollutants of emerging concern as they persist to reach non-target aquatic life and humans, the latter if it remains in treated water. The present study has identified a Pseudomonas mendocina strain that completely degrades guanylurea. The genome was sequenced and the genes involved in guanylurea metabolism were identified in three widely separated genomic regions. This knowledge advances the idea that guanylurea is not a dead end product and will allow for bioinformatic identification of the relevant genes in wastewater treatment plant microbiomes and other environments subjected to metagenomic sequencing.

Metformin Contamination in Global Waters: Biotic and Abiotic Transformation, Byproduct Generation and Toxicity, and Evaluation as a Pharmaceutical Indicator

“…Transformation pathways and TPs of metformin during (A) wastewater treatment bioreactor, (B) advanced oxidation process (detailed information on the transformation process in pathways I and II is provided in Figure S2), (C) natural aquatic system, and (D) water chlorination process (the dotted arrow indicates that the pathway can occur but has yet to be identified). The figures are created based on information collected from published papers. ,,,,,,,,− , The major physical and chemical properties of these TPs are available in Table .…”

“…GU was considered the “dead-end” TP of metformin during aerobic treatments with a reported bioconversion rate of 86.2 ± 8.8%, whereas MBG only accounted for 0.12% of the initial metformin . However, Tassoulas et al found that the Pseudomonas mendocina strain GU isolated from the activated sludge of a WWTP in Saint Paul, Minnesota (in the U.S.) could hydrolyze GU into guanidine (CN 3 H 5 ), CO 2 , and NH 3 , and the generated guanidine further underwent carboxylation, deamination, and hydrolysis to be completely mineralized . Because proteins in urine and many other chemicals containing guanidine moieties may produce guanidine during aerobic treatment, the occurrence of guanidine in effluents cannot exclusively indicate GU transformation.…”

“…The varied GU/metformin values in different WWTPs likely result from differences in microbial communities and operation performance . However, a much higher concentration of GU in the influent of a German WWTP (up to 2100 μg/L) was found than could be accounted for by metformin concentration in the influent (highest level of 86 μg/L) (Table S3), indicating that GU may also derive from the transformation of other compounds containing a guanidine moiety (e.g., cyanoguanidine and guanidine disinfectants). , …”

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family

“…Such lines of inquiry are urgently important as bacteria continue to evolve around us. The Qac and Gdx subtypes, in particular, have found new roles in human-impacted environments, conferring bacterial resistance to household antiseptics ( 89 ), “dead-end” metabolites from the degradation of metformin ( 140 ) and other pharmaceuticals that accumulate in the environment or the human microbiome, and other agricultural and industrial chemicals ( 4 ). Genes encoding SMR transporters are currently spreading among bacterial populations, encountering new physiological contexts and substrate transport demands, driving co-selection of co-localized antimicrobial resistance genes in environmental reservoirs ( 76 ), and influencing microbial population compositions in the human microbiome and human-impacted environments ( 32 , 76 , 141 ), as the SMR transporters contribute to the ongoing story of natural selection at the human–microbe interface.…”

Mycoremediation of sewage sludge and manure with marine fungi for the removal of organic pollutants

“…Metformin is bioactive in many organisms changing the expression of different genes, including some responsible for endocrine pathways (Ambrosio-Albuquerque et al, 2021). Metformin is quite persistent and its degradation leads to another persistent compound, guanylurea, a "dead-end" compound in waste water treatment (Tassoulas et al, 2021). Synthetic hormones used for medication can have devastating effects on ecosystems even in the slightest concentration.…”