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 Ni²⁺-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.

双胍类药物二甲双胍是治疗 2 型糖尿病的一线降血糖药物，导致其出现在全球环境中。然而，人们对二甲双胍通过微生物分解代谢的命运知之甚少。在这里，我们表征了来自尿素水解酶超家族的 Ni ²⁺依赖性异六聚酶 (MetCaCb)，其催化二甲双胍水解成脒基脲和二甲胺。任一亚基单独时均无催化活性，但它们一起作为二甲双胍高度特异性的活性酶发挥作用。 MetCaCb复合物的晶体结构显示双核金属簇仅在MetCa中配位，MetCb作为其活性同源物的蛋白质结合剂。计算机模拟搜索和功能测定发现了一组在环境中表现出二甲双胍水解酶活性的 MetCaCb 样蛋白对。我们的研究结果不仅为二甲双胍分解代谢奠定了遗传和生化基础，而且还为古代酶对新出现的底物的适应提供了更多见解。