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

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Discovery of a Ni2+-dependent heterohexameric metformin hydrolase

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Li, Tao, Xu, Zhi-Jing, Zhang, Shu-Ting, Xu, Jia, Pan, Piaopiao, Zhou, Ning-Yi·2024·Nature Communications
DOI 10.1038/s41467-024-50409-7PMC PMC11271267

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Abstract

The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni 2+ -dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.

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Fig. 1 MetCa and MetCb together function as a Ni2+-dependent metformin hydrolase. a Schematic representation of metformin hydrolysis and the putative genes involved. DMA, dimethylamine. b Conversion of metformin by cell extracts of E. coli expressing MetCaCb alone or together with MetAB in the presence of various transition metal ions. The cell extracts were incubated with 10 mM metformin and 0.2 mM of the indicated metal ions, and guanylurea production was measured after 12 h of incubation. c Metformin hydrolase assays with purified MetCa, MetCb and co-purified MetCaCb. The components of the reaction mixtures are indicated below each column. The arginase inhibitor 2(S)-amino-6-boronohexonic acid (ABH) and the nickel (Ni)-specific chelator dimethylglyoxime (DMG) were tested for their effects on the activity of MetCaCb. All assays in (b) and (c) were performed in three independent preparations. Data are presented as mean values ± SD. d 13C-NMR spectra of 13C15N-labeled metformin (10 mM) reaction mixtures with or without recombinant MetCaCb. e Growth phenotypes of strain NyZ550 and its gene-knockout variants on 5 mM glucose (GLU), metformin (MET), and dimethylamine (DMA). The respective gene complementation and their growth phenotypes are indicated within parentheses. MET and DMA served as the sole sources of carbon and nitrogen, while GLU was used as the sole carbon source with ammonium sulfate as nitrogen source. The “+” means that the value of OD600nm was >0.1 at the stationary phase, which represents a more than 10-fold increase of the OD600nm. The “−” means there was no evident increase of the OD600nm observed at an initial inoculum of 0.01.
Fig. 1 MetCa and MetCb together function as a Ni2+-dependent metformin hydrolase. a Schematic representation of metformin hydrolysis and the putative genes involved. DMA, dimethylamine. b Conversion of metformin by cell extracts of E. coli expressing MetCaCb alone or together with MetAB in the presence of various transition metal ions. The cell extracts were incubated with 10 mM metformin and 0.2 mM of the indicated metal ions, and guanylurea production was measured after 12 h of incubation. c Metformin hydrolase assays with purified MetCa, MetCb and co-purified MetCaCb. The components of the reaction mixtures are indicated below each column. The arginase inhibitor 2(S)-amino-6-boronohexonic acid (ABH) and the nickel (Ni)-specific chelator dimethylglyoxime (DMG) were tested for their effects on the activity of MetCaCb. All assays in (b) and (c) were performed in three independent preparations. Data are presented as mean values ± SD. d 13C-NMR spectra of 13C15N-labeled metformin (10 mM) reaction mixtures with or without recombinant MetCaCb. e Growth phenotypes of strain NyZ550 and its gene-knockout variants on 5 mM glucose (GLU), metformin (MET), and dimethylamine (DMA). The respective gene complementation and their growth phenotypes are indicated within parentheses. MET and DMA served as the sole sources of carbon and nitrogen, while GLU was used as the sole carbon source with ammonium sulfate as nitrogen source. The “+” means that the value of OD600nm was >0.1 at the stationary phase, which represents a more than 10-fold increase of the OD600nm. The “−” means there was no evident increase of the OD600nm observed at an initial inoculum of 0.01.CC BY · PubMed Central
Fig. 2 Characterization of metformin hydrolase. a Co-purification analysis of MetCaCb. MBP-tagged MetCa with or without tag-free MetCb were expressed in E. coli cells and purified using a dextrin column. The asterisk indicates the position of MBP-tagged MetCa, and the triangle indicates the position of tag-free MetCb. The metformin hydrolase (MH) activity is indicated at the bottom. Uncropped image of the gel can be found in source data file. The result shown is from a representative experiment of more than three times. b The size exclusion chromatography (SEC) analysis of the His-tagged MetCaCb complex. The activities of each fraction were indicated by guanylurea production within 60 min in 1-mL reaction mixture containing 5 μl of enzyme, 20 mM metformin and 0.1 mM Ni2+. An active fraction was analyzed by SDS-PAGE (insert). The result shown is from a representative experiment of more than ten times. Uncropped image of the gel can be found in source data file. c Determination of the molecular weight of the active MetCaCb complex by SEC coupled with MALS. The red line represents the experimentally determined molecular weight. d Guanylurea production at various temperatures and pH 9.0. e Guanylurea production from metformin at various pH values. f Guanylurea production at various metformin concentrations was determined at pH 9.0 and pH 10.0. g Guanylurea production at various 1-methylbiguanide concentrations was determined at pH 9.0. All assays were performed in triplicate of the same preparation, and independent enzyme preparations exhibited consistent results. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
Fig. 2 Characterization of metformin hydrolase. a Co-purification analysis of MetCaCb. MBP-tagged MetCa with or without tag-free MetCb were expressed in E. coli cells and purified using a dextrin column. The asterisk indicates the position of MBP-tagged MetCa, and the triangle indicates the position of tag-free MetCb. The metformin hydrolase (MH) activity is indicated at the bottom. Uncropped image of the gel can be found in source data file. The result shown is from a representative experiment of more than three times. b The size exclusion chromatography (SEC) analysis of the His-tagged MetCaCb complex. The activities of each fraction were indicated by guanylurea production within 60 min in 1-mL reaction mixture containing 5 μl of enzyme, 20 mM metformin and 0.1 mM Ni2+. An active fraction was analyzed by SDS-PAGE (insert). The result shown is from a representative experiment of more than ten times. Uncropped image of the gel can be found in source data file. c Determination of the molecular weight of the active MetCaCb complex by SEC coupled with MALS. The red line represents the experimentally determined molecular weight. d Guanylurea production at various temperatures and pH 9.0. e Guanylurea production from metformin at various pH values. f Guanylurea production at various metformin concentrations was determined at pH 9.0 and pH 10.0. g Guanylurea production at various 1-methylbiguanide concentrations was determined at pH 9.0. All assays were performed in triplicate of the same preparation, and independent enzyme preparations exhibited consistent results. Data are presented as mean values ± SD. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 Structural features of MetCaCb. a The heterohexameric MetCaCb complex structure shown in ribbon (MetCa) and surface (MetCb) diagrams. Metal ions in the active sites are shown as green spheres. b Structural comparison of MetCa and MetCb. The atomic structures of α1 (claret) and β1(cyan) subunits are overlayed. The differential loops of MetCa or MetCb are indicated by black arrows. c Superposition of the active site-regions of MetCa and MetCb. Dashed black lines indicate metal coordination. d The conserved metal coordinating residues in representative sequences of arginase family proteins. Detailed information on the selected sequences can be found in the Methods section. Pos.: residue positions in MetCa. e The active site pocket in MetCa is capped by the N-terminal loop structure of MetCb. The residues around the entrance of pocket are shown as sticks. f Schematic drawing of the active site with a metformin molecule (purple stick) docked into it. The metal ion coordinating residues are colored in gray.
Fig. 3 Structural features of MetCaCb. a The heterohexameric MetCaCb complex structure shown in ribbon (MetCa) and surface (MetCb) diagrams. Metal ions in the active sites are shown as green spheres. b Structural comparison of MetCa and MetCb. The atomic structures of α1 (claret) and β1(cyan) subunits are overlayed. The differential loops of MetCa or MetCb are indicated by black arrows. c Superposition of the active site-regions of MetCa and MetCb. Dashed black lines indicate metal coordination. d The conserved metal coordinating residues in representative sequences of arginase family proteins. Detailed information on the selected sequences can be found in the Methods section. Pos.: residue positions in MetCa. e The active site pocket in MetCa is capped by the N-terminal loop structure of MetCb. The residues around the entrance of pocket are shown as sticks. f Schematic drawing of the active site with a metformin molecule (purple stick) docked into it. The metal ion coordinating residues are colored in gray.CC BY · PubMed Central
Fig. 4 Bioinformatic and functional analyzes of MetCaCb-like paired arginase family proteins. a Phylogenetic tree of MetCaCb-like arginase family protein pairs. The six conserved metal ion coordinating residues for each sequence are shown on the right side, and their positions in MetCa are indicated on the top of the columns. The residues with a cyan background are mutated residues, differing from the conserved residues marked with a pink background. Human arginase I (hARGI) was used as an outgroup. b MetCaCb-like protein pairs formed hexamers. The method for co-purification of AcMetCaCb, HmMetCaCb, and RsMetCaCb was similar to that for MetCaCb. Notably, elution peaks at a volume of ~12.9 mL, indicative of hexameric protein complexs, were selected for subsequent enzymatic activity tests. Purification of HmMetCaCb was accompanied by an inactive peak (15.9 mL) which corresponded to the HmMetCa monomer. c Assessment of the metformin hydrolase activity of the MetCaCb-like protein pairs. The data represent the mean values of two replicates using independent enzyme preparation. Source data are provided as a Source Data file.
Fig. 4 Bioinformatic and functional analyzes of MetCaCb-like paired arginase family proteins. a Phylogenetic tree of MetCaCb-like arginase family protein pairs. The six conserved metal ion coordinating residues for each sequence are shown on the right side, and their positions in MetCa are indicated on the top of the columns. The residues with a cyan background are mutated residues, differing from the conserved residues marked with a pink background. Human arginase I (hARGI) was used as an outgroup. b MetCaCb-like protein pairs formed hexamers. The method for co-purification of AcMetCaCb, HmMetCaCb, and RsMetCaCb was similar to that for MetCaCb. Notably, elution peaks at a volume of ~12.9 mL, indicative of hexameric protein complexs, were selected for subsequent enzymatic activity tests. Purification of HmMetCaCb was accompanied by an inactive peak (15.9 mL) which corresponded to the HmMetCa monomer. c Assessment of the metformin hydrolase activity of the MetCaCb-like protein pairs. The data represent the mean values of two replicates using independent enzyme preparation. Source data are provided as a Source Data file.CC BY · PubMed Central

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Research works printed but not linked: 48

  1. Dos, S. G. R. et al. Medicines as an emergent contaminant: the review of microbial biodegration potential. Folia Microbiol. (Praha) 67, 157–174 (2022). · DOI
  2. Wilkinson, J. L. et al. Pharmaceutical pollution of the world’s rivers. Proc. Natl Acad. Sci. USA 119, e2113947119 (2022). · DOI
  3. Lindell, A. E., Zimmermann-Kogadeeva, M. & Patil, K. R. Multimodal interactions of drugs, natural compounds and pollutants with the gut microbiota. Nat. Rev. Microbiol. 20, 431–443 (2022). · DOI
  4. Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R. & Goodman, A. L. Mapping human microbiome drug metabolism by gut bacteria and their genes. Nature 570, 462–467 (2019). · DOI · Paper page
  5. Werner, E. A. & Bell, J. CCXIV.—The preparation of methylguanidine, and of ββ-dimethylguanidine by the interaction of dicyanodiamide, and methylammonium and dimethylammonium chlorides respectively. J. Chem. Soc. Trans. 121, 1790–1794 (1922). · DOI
  6. Foretz, M., Guigas, B. & Viollet, B. Metformin: update on mechanisms of action and repurposing potential. Nat. Rev. Endocrinol. 19, 460–476 (2023). · DOI
  7. Balakrishnan, A., Sillanpää, M., Jacob, M. M. & Vo, D. N. Metformin as an emerging concern in wastewater: Occurrence, analysis and treatment methods. Environ. Res. 213, 113613 (2022). · DOI
  8. Elizalde-Velazquez, G. A. & Gomez-Olivan, L. M. Occurrence, toxic effects and removal of metformin in the aquatic environments in the world: Recent trends and perspectives. Sci. Total Environ. 702, 134924 (2020). · DOI
  9. Cabreiro, F. et al. Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism. Cell 153, 228–239 (2013). · DOI · Paper page
  10. Forslund, K. et al. Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. Nature 528, 262–266 (2015). · DOI · Paper page
  11. He, Y., Zhang, Y. & Ju, F. Metformin contamination in global waters: biotic and abiotic transformation, byproduct generation and toxicity, and evaluation as a pharmaceutical indicator. Environ. Sci. Technol. 56, 13528–13545 (2022). · DOI
  12. Briones, R. M., Zhuang, W. Q. & Sarmah, A. K. Biodegradation of metformin and guanylurea by aerobic cultures enriched from sludge. Environ. Pollut. 243, 255–262 (2018). · DOI
  13. Poursat, B. A. J. et al. Biodegradation of metformin and its transformation product, guanylurea, by natural and exposed microbial communities. Ecotox. Environ. Saf. 182, 109414 (2019). · DOI
  14. Tassoulas, L. J., Robinson, A., Martinez-Vaz, B., Aukema, K. G. & Wackett, L. P. Filling in the gaps in metformin biodegradation: a new enzyme and a metabolic pathway for guanylurea. Appl. Environ. Microbiol. 87, e03003–e03020 (2021). · DOI
  15. Martinez-Vaz, B. M. et al. Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products. Front. Bioeng. Biotechnol. 10, 1086261 (2022). · DOI
  16. Li, T., Xu, Z.-J. & Zhou, N.-Y. Aerobic Degradation of the antidiabetic drug metformin by Aminobacter sp. strain NyZ550. Environ. Sci. Technol. 57, 1510–1519 (2023). · DOI
  17. Chaignaud, P. et al. A Methylotrophic bacterium growing with the antidiabetic drug metformin as its sole carbon, nitrogen and energy source. Microorganisms 10, 2302 (2022). · DOI
  18. Hillmann, K. B. & Niehaus, T. D. Genome sequences of two Pseudomonas isolates that can use metformin as the sole nitrogen source. Microbiol. Resour. Announc. 11, e0063922 (2022). · DOI
  19. Hernández, V. M., Arteaga, A. & Dunn, M. F. Diversity, properties and functions of bacterial arginases. FEMS Microbiol. Rev. 45, fuab034 (2021). · DOI
  20. Baggio, R. et al. Inhibition of Mn2+2-arginase by borate leads to the design of a transition state analogue inhibitor, 2(S)-amino-6-boronohexanoic acid. J. Am. Chem. Soc. 119, 8107–8108 (1997). · DOI
  21. Di Costanzo, L. et al. Crystal structure of human arginase I at 1.29-Å resolution and exploration of inhibition in the immune response. Proc. Natl Acad. Sci. USA 102, 13058–13063 (2005). · DOI
  22. Velázquez-Libera, J. L., Caballero, J., Tuñón, I., Hernández-Rodríguez, E. W. & Ruiz-Pernía, J. J. On the nature of the enzyme–substrate complex and the reaction mechanism in human arginase I. A combined molecular dynamics and QM/MM study. ACS Catal. 10, 8321–8333 (2020). · DOI
  23. O’Leary, N. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 44, D733–D745 (2015). · DOI · Paper page
  24. Tassoulas, L. J., Rankin, J. A., Elias, M. H. & Wackett, L. P. Dinickel enzyme evolved to metabolize the pharmaceuticalmetformin and its implications for wastewater and human microbiomes. Proc. Natl Acad. Sci. USA 121, e2312652121 (2024). · DOI
  25. Miller, D., Xu, H. & White, R. H. A new subfamily of agmatinases present in Methanogenic Archaea Is Fe(II) dependent. Biochemistry 51, 3067–3078 (2012). · DOI
  26. Saragadam, T., Kumar, S. & Punekar, N. Characterization of 4-guanidinobutyrase from Aspergillus niger. Microbiology 165, 396–410 (2019). · DOI
  27. Arakawa, N., Igarashi, M., Kazuoka, T., Oikawa, T. & Soda, K. D-arginase of Arthrobacter sp. KUJ 8602: characterization and its identity with Zn2+-guanidinobutyrase. J. biochem. 133, 33–42 (2003). · DOI
  28. Funck, D. et al. Discovery of a Ni2+-dependent guanidine hydrolase in bacteria. Nature 603, 515–521 (2022). · DOI
  29. Romero, N. et al. Mammalian agmatinases constitute unusual members in the family of Mn2+-dependent ureahydrolases. J. Inorg. Biochem. 166, 122–125 (2017). · DOI
  30. Hai, Y., Kerkhoven, E. J., Barrett, M. P. & Christianson, D. W. Crystal structure of an arginase-like protein from Trypanosoma brucei that evolved without a binuclear manganese cluster. Biochemistry 54, 458–471 (2015). · DOI
  31. Mothersole, R. G. et al. Sequence divergence in the arginase domain of ornithine decarboxylase/arginase in Fusobacteriacea leads to loss of function in oral associated species. Biochemistry 61, 1378–1391 (2022). · DOI
  32. Garcia, D., Uribe, E., Salgado, M. & Martínez, M. Mutagenic and kinetic support for an allosteric site in arginase from the extreme thermophile Bacillus caldovelox, which allows activation by arginine. Biochimie. 108, 8–12 (2014). · DOI
  33. Lobos, M. et al. Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I. J. Struct. Biol. 211, 107533 (2020). · DOI
  34. Lavulo, L. et al. Subunit-subunit interactions in trimeric arginase. Generation of active monomers by mutation of a single amino acid. J. biol. Chem. 276, 14242–14248 (2001). · DOI
  35. Adrain, C. & Freeman, M. New lives for old: evolution of pseudoenzyme function illustrated by iRhoms. Nat. rev. Mol. cell biol. 13, 489–498 (2012). · DOI
  36. Schneider, N. O. et al. Solving the conundrum: widespread proteins annotated for urea metabolism in bacteria are carboxyguanidine deiminases mediating nitrogen assimilation from guanidine. Biochemistry 59, 3258–3270 (2020). · DOI
  37. Fida, T. T., Palamuru, S., Pandey, G. & Spain, J. C. Aerobic biodegradation of 2,4-dinitroanisole by Nocardioides sp. strain JS1661. Appl. Environ. Microbiol. 80, 7725–7731 (2014). · DOI
  38. Balaich, J. et al. The human microbiome encodes resistance to the antidiabetic drug acarbose. Nature 600, 110–115 (2021). · DOI
  39. Tian, J. et al. Inactivation of the antidiabetic drug acarbose by human intestinal microbial-mediated degradation. Nat. Metab. 5, 1–14 (2023). · DOI
  40. Brown, J. B., Conner, C. & Nichols, G. A. Secondary failure of metformin monotherapy in clinical practice. Diabetes Care 33, 501–506 (2010). · DOI
  41. Liu, H., Wang, S. J. & Zhou, N. Y. A new isolate of Pseudomonas stutzeri that degrades 2-chloronitrobenzene. Biotechnol. Lett. 27, 275–278 (2005). · DOI
  42. Kabsch, W. XDS. Acta Crystallogr D. Biol. Crystallogr 66, 125–132 (2010). · DOI · Paper page
  43. McCoy, A. et al. PHASER crystallographic software. J. appl. Crystallogr. 40, 658–674 (2007). · DOI · Paper page
  44. Adams, P. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011). · DOI
  45. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. Sect. D., Biol. Crystallogr. 66, 486–501 (2010). · DOI · Paper page
  46. Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018). · DOI · Paper page
  47. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010). · DOI · Paper page
  48. Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic acids res 50, W276–W279 (2022). · DOI

Metadata

Authors
Li, T., Xu, Z.-J., Zhang, S.-T., Xu, J., Pan, P., & Zhou, N.-Y.
Affiliations
Shanghai Jiao Tong University (CN)
Corresponding author
Zhou, Ning-Yi
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-07-20
Volume
15
Issue
1
Pages
6121-6121
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-50409-7
PMC
PMC11271267
OpenAlex
W4400842703
Cited by
13
References
2
Citations by year
2026: 4 · 2025: 5 · 2024: 4
FWCI
1.30
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)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Natural Science Foundation of China (32230001)
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
metformin, nickel-dependent enzymes, guanylurea, dimethylamine, type 2 diabetes drugs, environmental fate of pharmaceuticals, enzyme evolution, crystal structure
MeSH terms
Bacterial Proteins, Hydrolases, Hydrolysis, Metformin, Models, Molecular, Nickel, Substrate Specificity, Crystallography, X-Ray

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