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Cited by 13Pharmaceutical and Antibiotic Environmental ImpactsEnzyme Structure and FunctionEnzyme Catalysis and Immobilization

The DOI matches its Crossref record; 48 of the 48 references it cites were found in a registry or an index.

Metformin hydrolase is a recently evolved nickel-dependent heteromeric ureohydrolase

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Sinn, M., Riede, L., Fleming, J. R., Funck, D., Lutz, H., Bachmann, A., Mayans, O., Hartig, J. S.·2024·Nature Communications
DOI 10.1038/s41467-024-51752-5PMC PMC11399263

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Abstract

The anti-diabetic drug metformin is one of the most widely prescribed medicines in the world. Together with its degradation product guanylurea, it is a major pharmaceutical pollutant in wastewater treatment plants and surface waters. An operon comprising two genes of the ureohydrolase family in Pseudomonas and Aminobacter species has recently been implicated in metformin degradation. However, the corresponding proteins have not been characterized. Here we show that these genes encode a Ni 2+ -dependent enzyme that efficiently and specifically hydrolyzes metformin to guanylurea and dimethylamine. The active enzyme is a heteromeric complex of α- and β- subunits in which only the α-subunits contain the conserved His and Asp residues for the coordination of two Ni 2+ ions in the active site. A crystal structure of metformin hydrolase reveals an α 2 β 4 stoichiometry of the hexameric complex, which is unprecedented in the ureohydrolase family. By studying a closely related but more widely distributed enzyme, we find that the putative predecessor specifically hydrolyzes dimethylguanidine instead of metformin. Our findings establish the molecular basis for metformin hydrolysis to guanylurea as the primary pathway for metformin biodegradation and provide insight into the recent evolution of ureohydrolase family proteins in response to an anthropogenic compound.

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Figures

Fig. 1 Purification and activities of recombinant metformin and dimethylguanidine hydrolases MefH and DmgH. A Genomic structure of the genes conferring metformin or dimethylguanidine hydrolase activity. B Reactions catalyzed by heteromeric metformin hydrolase MefH and dimethylguanidine hydrolase DmgH. C Representative Coomassie stained denaturing polyacrylamide gel of DmgH, A. niigataenis AnMefH and P. mendocina PmMefH purified by Strep-Tactin affinity chromatography after expression in the presence of 0.5 mM Ni2+. In all three expression systems, the first hydrolase was Strep-tagged and the second hydrolase was co-purified, indicating the formation of a heteromeric complex. D Bar chart showing the specific activities with 2 mM metformin or dimethylguanidine as substrates of the three different enzyme systems. Activities were determined as the substrate and enzyme-dependent production of urea (U) or guanylurea (GU). Guanylurea was determined by LC-MS and urea was determined by a colorimetric assay. E Metal dependency of PmMefH. PmMefH was expressed in the presence of 50 µM of the respective metal ions and purified by nickel affinity chromatography (Supplementary Fig. 5). Guanylurea production rates were determined for purified PmMefH expressed in media supplemented with the divalent cations of the indicated metals. F Urea (U) or guanylurea (GU) production at different substrate concentrations was measured to determine Michaelis constant KM and maximal reaction rate (vmax) for DmgH (black circles), AnMefH (green squares) and PmMefH (blue triangles). Data were fitted using the Michaelis-Menten equation (R² = 0.9934; R² = 0.9946, R² = 0.9950, respectively). G Urea (U) or guanylurea (GU) production was measured at different temperatures to determine the apparent temperature optimum of MefH and DmgH. Legend as in E. For D–G, the data represent the average of triplicates and consistent results were obtained with independent preparations (n = 3, error bars, s.d.). Please note that for the temperature dependency of DmgH in G the mean of duplicates are shown and some error bars are not visible in F and G as they are smaller than the symbols used to indicate the means. Source data are provided as a Source Data file.
Fig. 1 Purification and activities of recombinant metformin and dimethylguanidine hydrolases MefH and DmgH. A Genomic structure of the genes conferring metformin or dimethylguanidine hydrolase activity. B Reactions catalyzed by heteromeric metformin hydrolase MefH and dimethylguanidine hydrolase DmgH. C Representative Coomassie stained denaturing polyacrylamide gel of DmgH, A. niigataenis AnMefH and P. mendocina PmMefH purified by Strep-Tactin affinity chromatography after expression in the presence of 0.5 mM Ni2+. In all three expression systems, the first hydrolase was Strep-tagged and the second hydrolase was co-purified, indicating the formation of a heteromeric complex. D Bar chart showing the specific activities with 2 mM metformin or dimethylguanidine as substrates of the three different enzyme systems. Activities were determined as the substrate and enzyme-dependent production of urea (U) or guanylurea (GU). Guanylurea was determined by LC-MS and urea was determined by a colorimetric assay. E Metal dependency of PmMefH. PmMefH was expressed in the presence of 50 µM of the respective metal ions and purified by nickel affinity chromatography (Supplementary Fig. 5). Guanylurea production rates were determined for purified PmMefH expressed in media supplemented with the divalent cations of the indicated metals. F Urea (U) or guanylurea (GU) production at different substrate concentrations was measured to determine Michaelis constant KM and maximal reaction rate (vmax) for DmgH (black circles), AnMefH (green squares) and PmMefH (blue triangles). Data were fitted using the Michaelis-Menten equation (R² = 0.9934; R² = 0.9946, R² = 0.9950, respectively). G Urea (U) or guanylurea (GU) production was measured at different temperatures to determine the apparent temperature optimum of MefH and DmgH. Legend as in E. For D–G, the data represent the average of triplicates and consistent results were obtained with independent preparations (n = 3, error bars, s.d.). Please note that for the temperature dependency of DmgH in G the mean of duplicates are shown and some error bars are not visible in F and G as they are smaller than the symbols used to indicate the means. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 Crystal structure of MefH. Global view of the MefH crystal structure. MefH is a dimer of two heterotrimers. Green spheres depict the Ni2+ ions in the active site of the MefHα subunits. A The heterotrimer is formed by the active MefHα subunit (red) and two inactive MefHβ subunits (blue). B The MefHα subunits interact tightly whereas between the loosely interacting MefHβ subunits a cleft is formed. C Overlay of a MefHα (red) and a MefHβ (purple) subunit with bound Ni2+ (green spheres) and urea (beige). D Close-up of the active site of MefHα with the side chains of the nickel-coordinating amino acids and the corresponding amino acids of the degenerated binding site of MefHβ, where no metal was bound. For visual clarity, biuret bound at the N-termini of the MefHα subunits is not displayed. Ligand binding is further illustrated in Supplementary Figs. 10, 11. Model coordinates and experimental diffraction data have been deposited with the Protein Data Bank under accession code 8RYI.
Fig. 2 Crystal structure of MefH. Global view of the MefH crystal structure. MefH is a dimer of two heterotrimers. Green spheres depict the Ni2+ ions in the active site of the MefHα subunits. A The heterotrimer is formed by the active MefHα subunit (red) and two inactive MefHβ subunits (blue). B The MefHα subunits interact tightly whereas between the loosely interacting MefHβ subunits a cleft is formed. C Overlay of a MefHα (red) and a MefHβ (purple) subunit with bound Ni2+ (green spheres) and urea (beige). D Close-up of the active site of MefHα with the side chains of the nickel-coordinating amino acids and the corresponding amino acids of the degenerated binding site of MefHβ, where no metal was bound. For visual clarity, biuret bound at the N-termini of the MefHα subunits is not displayed. Ligand binding is further illustrated in Supplementary Figs. 10, 11. Model coordinates and experimental diffraction data have been deposited with the Protein Data Bank under accession code 8RYI.CC BY · PubMed Central
Fig. 3 Investigation of substrate specificity determinants. A Overlay of the active sites of the AnMefH crystal structure (gray) and an AlphaFold model of DmgH (green). The side chains of the metal binding residues of AnMefH are shown (gray). These amino acids are identical in DmgH and MefH. Residues that were exchanged in DmgH for the corresponding residues of MefH are highlighted in blue, if substrate specificity was not changed, or red, if substrate specificity was changed. B Specific activities for the combinations of DmgH and AnMefH subunits and different variants of DmgH with 2 mM dimethylguanidine or metformin. Data represent the average ± s.d. of triplicates and consistent results were obtained with independent preparations (n = 3, error, s.d.). n.d. not detected. Source data are provided as a Source Data file.
Fig. 3 Investigation of substrate specificity determinants. A Overlay of the active sites of the AnMefH crystal structure (gray) and an AlphaFold model of DmgH (green). The side chains of the metal binding residues of AnMefH are shown (gray). These amino acids are identical in DmgH and MefH. Residues that were exchanged in DmgH for the corresponding residues of MefH are highlighted in blue, if substrate specificity was not changed, or red, if substrate specificity was changed. B Specific activities for the combinations of DmgH and AnMefH subunits and different variants of DmgH with 2 mM dimethylguanidine or metformin. Data represent the average ± s.d. of triplicates and consistent results were obtained with independent preparations (n = 3, error, s.d.). n.d. not detected. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Growth of A. niigataensis on different carbon and nitrogen sources. A. niigataensis DSM7050 was grown at 30 °C in a minimal medium containing 10 mM ammonium and 0.5% (w/v) glycerol (ctrl.), 10 mM dimethylguanidine (dmgd) or 10 mM metformin (mef) as carbon, nitrogen, and energy source. OD600 was measured to assess bacterial growth. Data points during exponential phase were fitted with an exponential equation to calculate the doubling time tD. Data are from three independent cultures. Source data are provided as a Source Data file.
Fig. 4 Growth of A. niigataensis on different carbon and nitrogen sources. A. niigataensis DSM7050 was grown at 30 °C in a minimal medium containing 10 mM ammonium and 0.5% (w/v) glycerol (ctrl.), 10 mM dimethylguanidine (dmgd) or 10 mM metformin (mef) as carbon, nitrogen, and energy source. OD600 was measured to assess bacterial growth. Data points during exponential phase were fitted with an exponential equation to calculate the doubling time tD. Data are from three independent cultures. Source data are provided as a Source Data file.CC BY · PubMed Central

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Cites

Research works printed but not linked: 2

  1. Urakami, T., Araki, H., Oyanagi, H., Suzuki, K.-I. & Komagata, K. Transfer of Pseudomonas aminovorans (den Dooren de Jong 1926) to Aminobacter gen. nov. as Aminobacter aminovorans comb. nov. and Description of Aminobacter aganoensis sp. nov. and Aminobacter niigataensis sp. nov. Int. J. Syst. Evol. Microbiol. 42, 84–92 (1992).
  2. Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98 (1999).

Metadata

Authors
Sinn, M., Riede, L., Fleming, J. R., Funck, D., Lutz, H., Bachmann, A., Mayans, O., & Hartig, J. S.
Affiliations
University of Konstanz (DE)
Corresponding author
  • Sinn, M.
  • Hartig, J. S.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-09-14
Volume
15
Issue
1
Pages
8045-8045
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-51752-5
PMC
PMC11399263
OpenAlex
W4402503221
Cited by
13
References
48
Citations by year
2026: 6 · 2025: 4 · 2024: 3
FWCI
1.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Desy publication database (The Deutsches Elektronen-Synchrotron) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • KOPS (University of Konstanz) (submittedVersion, cc-by)
  • DESY Publication Database (PUBDB) (Deutsches Elektronen-Synchrotron)
Funders
Universität Konstanz
Topics
  • Physical Sciences › Environmental Science › Pollution › Pharmaceutical and Antibiotic Environmental Impacts
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
guanylurea, nickel-dependent enzymes, enzyme evolution, Pseudomonas, Aminobacter, crystal structure, pharmaceutical pollutants, wastewater treatment, enzyme active site, dimethylamine
MeSH terms
Bacterial Proteins, Biodegradation, Environmental, Hydrolysis, Metformin, Models, Molecular, Nickel, Pseudomonas, Ureohydrolases, Crystallography, X-Ray, Catalytic Domain

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