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

Abstract

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 K_M 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. 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.

Keywords: biodegradation; human gut microbiome; metallohydrolase; metformin; nickel.

Fig. 1.

Revealing the function of genes encoding metformin degradation in a Pseudomonas species. (A) Metformin-related metabolic genes in Pseudomonas mendocina sp. MET-2 are in a putative operon which encode metformin hydrolase (MfmA and MfmB), nickel metabolism proteins (HypB and HypA), a putative nickel importer (UreJ), and a putative metformin transporter (CodB). MfmA and MfmB are both homologs of the ureohydrolase superfamily and share 34% sequence identity to each other. The Pseudomonas species has additional genes to completely mineralize guanylurea as a nitrogen source and utilize dimethylamine as a source of carbon or nitrogen. NCBI Genbank identifiers for the mfmA and mfmB genes are shown above the genes in the putative operon. (B) Activity on metformin was found only in E. coli lysates when MfmA and MfmB were coexpressed (MfmAB) and not individually. Lysates were incubated with 1 mM metformin for 1 h in 20 mM CHES pH 9 with 1 mM NiCl₂ before being sampled using an HPLC method that can separate guanylurea from metformin. Guanylurea was identified as the reaction product of MfmAB. (C) MfmB copurifies with His-tagged MfmA. Stained, denaturing, polyacrylamide gel with purified MfmAB after IMAC. A band was observed for 6×His-tagged MfmA and a more intense band was seen for MfmB despite not being tagged. See SI Appendix, Fig. S2 for the full gel. (D) Identification of dimethylamine as a reaction product of MfmAB by ¹H NMR. NMR spectra were obtained for 50 mM metformin in 50 mM ammonium formate, 200 mM NaCl pH 8.5, and 1 mM NiCl₂ with 20% (v/v) D₂O before (blue spectrum) and after 1-h incubation with 200 μg purified MfmAB (red spectrum). The major shift for the methyl hydrogens changed upon MfmAB addition from 3.06 ppm to 2.73 ppm which was identical to the shift found for the dimethylamine standard. See SI Appendix, Fig. S2, for full NMR spectra. (E) MfmA and MfmB are homologous to arginase and agmatinase with binuclear divalent metal centers, yet MfmB has lost several key metal binding residues. The active site of MfmA is shown with the binuclear metal center and the several histidine and aspartate residues binding it. A multiple sequence alignment showing sequence conservation shared between human arginase I (PDB 2AEB), E. coli agmatinase (PDB 7LOL), MfmA, and MfmB is depicted. Numbering of amino acids is based on the sequence of MfmA.

Fig. 2.

MfmAB is Ni²⁺-dependent and shows exquisite specificity for metformin. (A) pH-activity dependence of MfmAB. Enzyme was incubated in different pH buffers for 15 min before adding 15 mM metformin and sampling after another 15 min. The pH buffer types used, 50 mM, were PIPES (pH 6 to 7, shown as circles), HEPES (pH 7 to 8.5, squares), CHES (pH 8.5 to 10, triangles), and CAPS (pH 10 to 11, pentagons). Error bars denote one SD of the mean from averaging two technical replicates. (B) Metal-activity dependence of MfmAB. Enzyme, stripped of metal, was reconstituted with or without 0.1 mM of several divalent metals, and activity was measured by a coupled-enzyme assay in 50 mM CHES pH 9 with 5 mM metformin. Error bars denote one SD of the mean from two technical replicates. (C) Plot of specificity ratios of MfmAB activity for various substrates relative to metformin. No other substrate tested showed activity more than 1% than that of metformin. Specific activities were measured by incubating purified MfmAB with 50 mM substrate in 100 mM CHES pH 9 buffer using a coupled-enzyme assay with two technical replicates. (D) Michaelis–Menten kinetics of metformin hydrolase at pH 9 (squares) and pH 8 (circles). Activity at several metformin concentrations was measured by observing guanylurea release using a coupled-enzyme assay in either 50 mM CHES pH 9 or 50 mM HEPES pH 8 buffer. Error bars denote one SD of the mean from three biological replicates. Black lines show the fit to the Michaelis–Menten equation.

Fig. 3.

Crystal structure of the MfmAB complex. (A) MfmAB is a heterotrimer with one subunit of MfmA per two subunits of MfmB (PDB 8SNF). (B) Overlay of MfmA and MfmB subunits shows high structural conservation apart from their N-terminal loops which provide contacts at the intersubunit interfaces of MfmAB (PDB 8SNF). (C) Active site of MfmA showing the anomalous dispersion difference maps contoured at 4σ found from diffraction data collected at the Ni edge, 8,347 eV, shown in orange mesh. Data were also collected under the Ni K-edge (8,250 eV), but the map (red mesh) shows no density peak at this contour level (PDB 8SNF). (D) Docking model of metformin bound to the active site of MfmA and mutagenesis. Select residues in the MfmA active site were substituted, singly, with the amino acids written in red next to the residue position and the relative specific activity to the WT stated above each residue tested. The residue substitution D188N was critical to activity which is implicated in activating the water that attacks the substrate. The relative activities reported are an average from two technical replicates with percent error of one SD being less than 3%. (E) Cavity of MfmA active site with metformin docked. (F) Overlay of docked metformin in MfmA with human arginase I (hARG1) bound with a boronic acid arginine analog, ABH, (PDB 2AEB). The docking model aligns well the dimethylamine moiety with the boronic acid group. (G) Overlay of metal centers from different ureohydrolase homologs, MfmA (PDB 8SNF), hARG1 (PDB 2AEB), E. coli SpeB (PDB 7LOL) and GdmH (PDB 7OI1). The positioning of the metals in MfmA (green), relative to the other homologs in the superfamily, may dictate substrate specificity.