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

Lambros J. Tassoulas, Ashley Robinson, Betsy Martinez-Vaz, Kelly G. Aukema, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

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, and the enzyme was purified and characterized. Guanylurea hydrolase, a newly described enzyme, was shown to transform guanylurea to one equivalent (each) 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, which 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 nontarget 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.

Original languageEnglish (US)
Article numbere03003-20
Pages (from-to)1-13
Number of pages13
JournalApplied and environmental microbiology
Volume87
Issue number11
DOIs
StatePublished - May 11 2021

Bibliographical note

Funding Information:
We acknowledge the kind gift of CgdAB enzyme from Martin St. Maurice of Marquette University. We thank Sara Palacios for help with the initial guanylurea enrichment cultures. This work was funded by the National Institute of Food and Agriculture, Agricultural and Food Research Initiative Competitive Program, Ecosystem Services and Agro-Ecosystem Management, grant no. 2019-67019-29403 to L.P.W. and B.M.-V. and National Institutes of Health Biotechnology training grant (5T32GM008347-27) for L.J.T.

Funding Information:
This work was funded by the National Institute of Food and Agriculture, Agricultural and Food Research Initiative Competitive Program, Ecosystem Services and Agro-Ecosystem Management, grant no. 2019-67019-29403 to L.P.W. and B.M.-V. and National Institutes of Health Biotechnology training grant (5T32GM008347-27) for L.J.T.

Publisher Copyright:
© 2021 American Society for Microbiology. All Rights Reserved.

Keywords

  • CgdAB
  • Pseudomonas mendocina
  • biodegradation
  • enzyme
  • genome
  • guanidine
  • guanylurea hydrolase
  • metformin

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

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