Abstract

Metformin is a first-line antidiabetic medication for type 2 diabetes and a widely dispersed emerging pollutant in aquatic systems. During medication, its strong positive charge (pKa  =  12.4) under physiological conditions necessitates cellular uptake via non-specific cationic transporters, such as organic cation transporter 1 (OCT1) in humans. During bacterial biodegradation of metformin, it was also proposed that the transport of metformin into bacterial cells is a prerequisite. Despite progress in elucidating the biodegradation mechanism of metformin, the bacterial membrane transport systems involved remain largely unexplored. Here, we characterize a metformin transporter (MetT) from the metformin utilizer Aminobacter sp. strain NyZ550. Gene knockout and complementation experiments demonstrate that metT is essential for the growth of strain NyZ550 on metformin. Efficient bacterial degradation of metformin necessitates the presence of both the membrane transporter and the downstream catabolic enzyme. Phylogenetic analysis revealed that MetT is a member of the nucleobase cation symporter 1 (NCS1) family, but forms a separate clade distinct from previously characterized NCS1 members. It exhibits distinct polar localization within the cytoplasmic membrane, as evidenced by a MetT-green fluorescent protein fusion construct. Radiolabeled uptake assays using 14C-metformin revealed a substrate transport affinity (Km) of 15.90 ± 1.75 µM, which starkly contrasts with the millimolar-range Km of human OCT1. Structural modeling and site-directed mutagenesis revealed a substrate-binding cavity consisting of aromatic residues, likely facilitating substrate recognition through cation-π interactions.