Discovery of a unique Ni2+-dependent heterohexameric metformin hydrolase

The biguanide drug metformin is prescribed worldwide as a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the environment around the world. Despite mounting evidence indicating that metabolic interactions of metformin with environmental or intestinal microbes affect ecological and human health, little is known about the fate of metformin by microbial catabolism. Here, we characterized a Ni²⁺-dependent bacterial enzyme that catalyzes the hydrolysis of metformin to form guanylurea and dimethylamine. The metformin hydrolase MetCaCb is a heterohexamer with an uneven α₂β₄ stoichiometry. Both subunits are from the arginase protein family, members of which are typically homomultimers. 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 clearly shows the coordination of the binuclear metal cluster only in MetCa, following a geometry typical for arginase family enzymes. A unique pseudoenzyme MetCb, which evolved without a binuclear metal cluster, contributes to hydrolase activity as a protein binder of its active cognate. An in-silico search and functional assay led to the discovery of 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 new insights into the adaption of the ancient arginase family proteins toward newly occurred substrate.