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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 (mfmAandmfmB) 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 × 10³M⁻¹s⁻¹and K_Mfor 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 themfmABgenes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater. 

The non-heme Fe( ii ) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and l -Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the l -Arg hydroxylation activity. In this study, we show that the two l -Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe–O bond in the EFE·Fe( iii )·OO − ˙·2OG· l -Arg complex can switch the EFE reactivity between l -Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing l -Arg hydroxylation. 

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD⁺, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD⁺, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD⁺and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.