Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes

Tassoulas, Lambros J.; Rankin, Joel A.; Elias, Mikael H.; Wackett, Lawrence P.

doi:10.1073/pnas.2312652121

Title: Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes

Journal Article · 25 February 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.2312652121 · OSTI ID:2470223

^[1];

^[1]; Elias, Mikael H. ^[1]; Wackett, Lawrence P. ^[1]

University of Minnesota, Minneapolis, MN (United States); University of Minnesota, St. Paul, MN (United States)

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 (mfmA and mfmB) 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 (k_cat/K_M) of 9.6 × 10³ M^-1s^-1 and KM for 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. In conclusion, this study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); National Science Foundation (NSF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470223

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 10 Vol. 121; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

References (65)

Structure and Reactivity of a Highly Twisted Amide Yamada, Shinji Angewandte Chemie International Edition in English, Vol. 32, Issue 7 https://doi.org/10.1002/anie.199310831	journal	July 1993
Disposition of metformin (N,N‐dimethylbiguanide) in man Sirtori, Cesare R.; Franceschini, Guido; Galli-Kienle, Marzia Clinical Pharmacology & Therapeutics, Vol. 24, Issue 6 https://doi.org/10.1002/cpt1978246683	journal	December 1978
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility Morris, Garrett M.; Huey, Ruth; Lindstrom, William Journal of Computational Chemistry, Vol. 30, Issue 16 https://doi.org/10.1002/jcc.21256	journal	December 2009
Biguanides Nattrass, M.; Alberti, K. G. M. M. Diabetologia, Vol. 14, Issue 2 https://doi.org/10.1007/BF01263443	journal	February 1978
Evolution of the arginase fold and functional diversity Dowling, D. P.; Di Costanzo, L.; Gennadios, H. A. Cellular and Molecular Life Sciences, Vol. 65, Issue 13 https://doi.org/10.1007/s00018-008-7554-z	journal	March 2008
Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group Carvajal, Nelson; Orellana, Marı́a S.; Salas, Mónica Archives of Biochemistry and Biophysics, Vol. 430, Issue 2 https://doi.org/10.1016/j.abb.2004.07.005	journal	October 2004
Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks Gerlt, John A.; Bouvier, Jason T.; Davidson, Daniel B. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1854, Issue 8 https://doi.org/10.1016/j.bbapap.2015.04.015	journal	August 2015
The Human Gut Microbiome: From Association to Modulation Schmidt, Thomas S. B.; Raes, Jeroen; Bork, Peer Cell, Vol. 172, Issue 6 https://doi.org/10.1016/j.cell.2018.02.044	journal	March 2018
Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy Pryor, Rosina; Norvaisas, Povilas; Marinos, Georgios Cell, Vol. 178, Issue 6 https://doi.org/10.1016/j.cell.2019.08.003	journal	September 2019
Environmental risk assessment of metformin and its transformation product guanylurea. I. Environmental fate Straub, Jürg Oliver; Caldwell, Daniel J.; Davidson, Todd Chemosphere, Vol. 216 https://doi.org/10.1016/j.chemosphere.2018.10.036	journal	February 2019
Health impact assessment after Danio rerio long-term exposure to environmentally relevant concentrations of metformin and guanylurea Elizalde-Velázquez, Gustavo Axel; Herrera-Vázquez, Selene Elizabeth; Gómez-Oliván, Leobardo Manuel Chemosphere, Vol. 341 https://doi.org/10.1016/j.chemosphere.2023.140070	journal	November 2023
Metformin Action: Concentrations Matter He, Ling; Wondisford, Fredric E. Cell Metabolism, Vol. 21, Issue 2 https://doi.org/10.1016/j.cmet.2015.01.003	journal	February 2015
Biodegradation of metformin and its transformation product, guanylurea, by natural and exposed microbial communities Poursat, Baptiste A. J.; van Spanning, Rob J. M.; Braster, Martin Ecotoxicology and Environmental Safety, Vol. 182 https://doi.org/10.1016/j.ecoenv.2019.109414	journal	October 2019
Occurrence of the antidiabetic drug Metformin and its ultimate transformation product Guanylurea in several compartments of the aquatic cycle Trautwein, Christoph; Berset, Jean-Daniel; Wolschke, Hendrik Environment International, Vol. 70 https://doi.org/10.1016/j.envint.2014.05.008	journal	September 2014
Metformin chlorination byproducts in drinking water exhibit marked toxicities of a potential health concern Zhang, Runshuai; He, Yuanzhen; Yao, Luxia Environment International, Vol. 146 https://doi.org/10.1016/j.envint.2020.106244	journal	January 2021
Decipher enzymes from human microbiota for drug discovery and development Beliaeva, Mariia A.; Wilmanns, Matthias; Zimmermann, Michael Current Opinion in Structural Biology, Vol. 80 https://doi.org/10.1016/j.sbi.2023.102567	journal	June 2023
Occurrence and removal of chemicals of emerging concern in wastewater treatment plants and their impact on receiving water systems Golovko, Oksana; Örn, Stefan; Sörengård, Mattias Science of The Total Environment, Vol. 754 https://doi.org/10.1016/j.scitotenv.2020.142122	journal	February 2021
Occurrence and fate of the antidiabetic drug metformin and its metabolite guanylurea in the environment and during drinking water treatment Scheurer, Marco; Michel, Amandine; Brauch, Heinz-Jürgen Water Research, Vol. 46, Issue 15 https://doi.org/10.1016/j.watres.2012.06.019	journal	October 2012
Emerging nitrogenous disinfection byproducts: Transformation of the antidiabetic drug metformin during chlorine disinfection of water Armbruster, Dominic; Happel, Oliver; Scheurer, Marco Water Research, Vol. 79 https://doi.org/10.1016/j.watres.2015.04.020	journal	August 2015
Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine Schneider, Nicholas O.; Tassoulas, Lambros J.; Zeng, Danyun Biochemistry, Vol. 59, Issue 35 https://doi.org/10.1021/acs.biochem.0c00537	journal	August 2020
Metformin Contamination in Global Waters: Biotic and Abiotic Transformation, Byproduct Generation and Toxicity, and Evaluation as a Pharmaceutical Indicator He, Yuanzhen; Zhang, Yanyan; Ju, Feng Environmental Science & Technology, Vol. 56, Issue 19 https://doi.org/10.1021/acs.est.2c02495	journal	September 2022
Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations Barros, Susana; Alves, Nélson; Pinheiro, Marlene Environmental Science & Technology, Vol. 57, Issue 2 https://doi.org/10.1021/acs.est.2c05719	journal	December 2022
Aerobic Degradation of the Antidiabetic Drug Metformin by Aminobacter sp. Strain NyZ550 Li, Tao; Xu, Zhi-Jing; Zhou, Ning-Yi Environmental Science & Technology, Vol. 57, Issue 3 https://doi.org/10.1021/acs.est.2c07669	journal	January 2023
Metformin and Other Pharmaceuticals Widespread in Wadeable Streams of the Southeastern United States Bradley, Paul M.; Journey, Celeste A.; Button, Daniel T. Environmental Science & Technology Letters, Vol. 3, Issue 6 https://doi.org/10.1021/acs.estlett.6b00170	journal	May 2016
ZINC20—A Free Ultralarge-Scale Chemical Database for Ligand Discovery Irwin, John J.; Tang, Khanh G.; Young, Jennifer Journal of Chemical Information and Modeling, Vol. 60, Issue 12 https://doi.org/10.1021/acs.jcim.0c00675	journal	October 2020
AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings Eberhardt, Jerome; Santos-Martins, Diogo; Tillack, Andreas F. Journal of Chemical Information and Modeling, Vol. 61, Issue 8 https://doi.org/10.1021/acs.jcim.1c00203	journal	July 2021
Mutagenesis of Rat Liver Arginase Expressed in Escherichia coli: Role of Conserved Histidines Cavalli, R. Christopher; Burke, Carl J.; Soprano, Dianne Robert Biochemistry, Vol. 33, Issue 35 https://doi.org/10.1021/bi00201a012	journal	September 1994
The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters Bar-Even, Arren; Noor, Elad; Savir, Yonatan Biochemistry, Vol. 50, Issue 21 https://doi.org/10.1021/bi2002289	journal	May 2011
Specific Metal Recognition in Nickel Trafficking Higgins, Khadine A.; Carr, Carolyn E.; Maroney, Michael J. Biochemistry, Vol. 51, Issue 40 https://doi.org/10.1021/bi300981m	journal	September 2012
Apo and Nickel-Bound Forms of the Pyrococcus horikoshii Species of the Metalloregulatory Protein: NikR Characterized by Molecular Dynamics Simulations Sindhikara, Daniel J.; Roitberg, Adrian E.; Merz, Kenneth M. Biochemistry, Vol. 48, Issue 50 https://doi.org/10.1021/bi9013352	journal	November 2009
l-Arginine Binding to Liver Arginase Requires Proton Transfer to Gateway Residue His141 and Coordination of the Guanidinium Group to the Dimanganese(II,II) Center Khangulov, Sergei V.; Sossong,, Thomas M.; Ash, David E. Biochemistry, Vol. 37, Issue 23 https://doi.org/10.1021/bi972874c	journal	May 1998
The Nonenzymatic Decomposition of Guanidines and Amidines Lewis, Charles A.; Wolfenden, Richard Journal of the American Chemical Society, Vol. 136, Issue 1 https://doi.org/10.1021/ja411927k	journal	December 2013
Order of Stability of Metal Complexes Irving, H.; Williams, R. J. P. Nature, Vol. 162, Issue 4123 https://doi.org/10.1038/162746a0	journal	November 1948
Erratum: Corrigendum: Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota Nature, Vol. 545, Issue 7652 https://doi.org/10.1038/nature22318	journal	May 2017
Metformin: update on mechanisms of action and repurposing potential Foretz, Marc; Guigas, Bruno; Viollet, Benoit Nature Reviews Endocrinology, Vol. 19, Issue 8 https://doi.org/10.1038/s41574-023-00833-4	journal	May 2023
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Discovery of a Ni2+-dependent guanidine hydrolase in bacteria Funck, D.; Sinn, M.; Fleming, J. R. Nature, Vol. 603, Issue 7901 https://doi.org/10.1038/s41586-022-04490-x	journal	March 2022
Microbial nickel proteins Kaluarachchi, Harini; Chan Chung, Kim C.; Zamble, Deborah B. Natural Product Reports, Vol. 27, Issue 5 https://doi.org/10.1039/b906688h	journal	January 2010
Occurrence of the antidiabetic drug metformin in sewage and surface waters in Germany Scheurer, Marco; Sacher, Frank; Brauch, Heinz-Jürgen Journal of Environmental Monitoring, Vol. 11, Issue 9 https://doi.org/10.1039/b909311g	journal	January 2009
Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling: A critical role for aspartate 153 Salas, Mónica; Rodríguez, Rolando; López, Nelia European Journal of Biochemistry, Vol. 269, Issue 22 https://doi.org/10.1046/j.1432-1033.2002.03255.x	journal	October 2002
Crystal structure of human arginase I at 1.29-A resolution and exploration of inhibition in the immune response Di Costanzo, L.; Sabio, G.; Mora, A. Proceedings of the National Academy of Sciences, Vol. 102, Issue 37 https://doi.org/10.1073/pnas.0504027102	journal	September 2005
Pharmaceutical pollution of the world’s rivers Wilkinson, John L.; Boxall, Alistair B. A.; Kolpin, Dana W. Proceedings of the National Academy of Sciences, Vol. 119, Issue 8 https://doi.org/10.1073/pnas.2113947119	journal	February 2022
Metformin acts in the gut and induces gut-liver crosstalk Tobar, Natália; Rocha, Guilherme Z.; Santos, Andrey Proceedings of the National Academy of Sciences, Vol. 120, Issue 4 https://doi.org/10.1073/pnas.2211933120	journal	January 2023
Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB Sydor, Andrew M.; Lebrette, Hugo; Ariyakumaran, Rishikesh Journal of Biological Chemistry, Vol. 289, Issue 7 https://doi.org/10.1074/jbc.M113.502781	journal	December 2013
Crystal Structure of Agmatinase Reveals Structural Conservation and Inhibition Mechanism of the Ureohydrolase Superfamily Ahn, Hyung Jun; Kim, Kyoung Hoon; Lee, Jiah Journal of Biological Chemistry, Vol. 279, Issue 48 https://doi.org/10.1074/jbc.M409246200	journal	September 2004
Phylogeny of related functions: the case of polyamine biosynthetic enzymes Sekowska, Agnieszka; Danchin, Antoine; Risler, Jean-Loup Microbiology, Vol. 146, Issue 8 https://doi.org/10.1099/00221287-146-8-1815	journal	August 2000
Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks Shannon, P. Genome Research, Vol. 13, Issue 11 https://doi.org/10.1101/gr.1239303	journal	November 2003
Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants Kabsch, W. Journal of Applied Crystallography, Vol. 26, Issue 6 https://doi.org/10.1107/S0021889893005588	journal	December 1993
MOLREP an Automated Program for Molecular Replacement Vagin, A.; Teplyakov, A. Journal of Applied Crystallography, Vol. 30, Issue 6, p. 1022-1025 https://doi.org/10.1107/S0021889897006766	journal	December 1997
Use of TLS parameters to model anisotropic displacements in macromolecular refinement Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Crystallographica Section D Biological Crystallography, Vol. 57, Issue 1 https://doi.org/10.1107/S0907444900014736	journal	January 2001
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/S0907444904019158	journal	November 2004
Solving structures of protein complexes by molecular replacement with Phaser McCoy, Airlie J. Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1 https://doi.org/10.1107/S0907444906045975	journal	December 2006
REFMAC 5 for the refinement of macromolecular crystal structures Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911001314	journal	March 2011
The journey of metformin from glycaemic control to mTOR inhibition and the suppression of tumour growth Amin, Sam; Lux, Andrew; O'Callaghan, Finbar British Journal of Clinical Pharmacology, Vol. 85, Issue 1 https://doi.org/10.1111/bcp.13780	journal	November 2018
Metformin disposition—A 40‐year‐old mystery Tucker, Geoffrey T.; Wesolowski, Carl A. British Journal of Clinical Pharmacology, Vol. 86, Issue 8 https://doi.org/10.1111/bcp.14320	journal	May 2020
Metformin kinetics in healthy subjects and in patients with diabetes mellitus. Tucker, Gt; Casey, C.; Phillips, Pj British Journal of Clinical Pharmacology, Vol. 12, Issue 2 https://doi.org/10.1111/j.1365-2125.1981.tb01206.x	journal	August 1981
The Effect of Intravenous Metformin on Glucose Metabolism During Hyperglycaemia in Type 2 Diabetes Sum, C. ‐F.; Webster, J. M.; Johnson, A. B. Diabetic Medicine, Vol. 9, Issue 1 https://doi.org/10.1111/j.1464-5491.1992.tb01716.x	journal	January 1992
Bioactive polyamine production by a novel hybrid system comprising multiple indigenous gut bacterial strategies Kitada, Yusuke; Muramatsu, Koji; Toju, Hirokazu Science Advances, Vol. 4, Issue 6 https://doi.org/10.1126/sciadv.aat0062	journal	June 2018
Filling in the Gaps in Metformin Biodegradation: a New Enzyme and a Metabolic Pathway for Guanylurea Tassoulas, Lambros J.; Robinson, Ashley; Martinez-Vaz, Betsy Applied and Environmental Microbiology, Vol. 87, Issue 11 https://doi.org/10.1128/AEM.03003-20	journal	May 2021
Genome Sequences of Two Pseudomonas Isolates That Can Use Metformin as the Sole Nitrogen Source Hillmann, Katie B.; Niehaus, Thomas D. Microbiology Resource Announcements, Vol. 11, Issue 9 https://doi.org/10.1128/mra.00639-22	journal	September 2022
Pharmacokinetics of metformin in the rat: assessment of the effect of hyperlipidemia and evidence for its metabolism to guanylurea Gabr, Raniah Q.; El-Sherbeni, Ahmed A.; Ben-Eltriki, Mohamed Canadian Journal of Physiology and Pharmacology, Vol. 95, Issue 5 https://doi.org/10.1139/cjpp-2016-0329	journal	May 2017
Towards a mechanistic understanding of reciprocal drug–microbiome interactions Zimmermann, Michael; Patil, Kiran Raosaheb; Typas, Athanasios Molecular Systems Biology, Vol. 17, Issue 3 https://doi.org/10.15252/msb.202010116	journal	March 2021
Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products Martinez-Vaz, Betsy M.; Dodge, Anthony G.; Lucero, Rachael M. Frontiers in Bioengineering and Biotechnology, Vol. 10 https://doi.org/10.3389/fbioe.2022.1086261	journal	December 2022
A Methylotrophic Bacterium Growing with the Antidiabetic Drug Metformin as Its Sole Carbon, Nitrogen and Energy Source Chaignaud, Pauline; Gruffaz, Christelle; Borreca, Adrien Microorganisms, Vol. 10, Issue 11 https://doi.org/10.3390/microorganisms10112302	journal	November 2022
Unexpected Resistance to Base-Catalyzed Hydrolysis of Nitrogen Pyramidal Amides Based on the 7-Azabicyclic[2.2.1]heptane Scaffold Ocampo Gutiérrez de Velasco, Diego Antonio; Su, Aoze; Zhai, Luhan Molecules, Vol. 23, Issue 9 https://doi.org/10.3390/molecules23092363	journal	September 2018

Similar Records

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

Journal Article · 2024 · Journal of General Physiology · OSTI ID:2283483

Lucero, Rachael M.; Demirer, Kemal; Yeh, Trevor Justin; +1 more

Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

Journal Article · 2013 · Biochemical and Biophysical Research Communications · OSTI ID:22210374

Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert; +1 more

Toxicity and toxicokinetics of metformin in rats

Journal Article · 2010 · Toxicology and Applied Pharmacology · OSTI ID:21344892

Quaile, Michael P; Melich, David H; Jordan, Holly L; +4 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
biochemistry
biodegradation
biological sciences
human gut microbiome
metallohydrolase
metformin
nickel
science & technology

Title: Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes

Citation Formats

References (65)

Similar Records

Related Subjects