Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

Abstract

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

On the link between antibiotic resistance, diabetes, and wastewater

“…The research provides a comprehensive analysis of the SMRGdx subtype, including biochemical, structural, and mechanistic attributes. These findings are relevant not only to other members of the SMR family but also to other multidrug transporters (MDTs), proteins best known for their role in microbial antibiotic resistance (this issue: Lucero et al, 2024 ).…”

“… Lucero et al (2024) solved a crystal structure of Gdx-Clo in the presence of guanylurea to investigate if it occupies the same binding site as guanidinium in Gdx-Clo. The crystal structure of Gdx-Clo with guanylurea was solved up to 2.1 Å, which showed that guanylurea occupies the same binding pocket as Gdm + in Gdx-Clo.…”

“…Metformin, a biguanide drug, is structurally similar to nitrogenous waste products such as guanidinium (Gdm + ) and guanylurea, which SMRGdx exports. The study conducted by Lucero et al (2024) used various methods to investigate how SMRGdx and other SMR proteins interact with metformin metabolites. These proteins are linked to horizontal gene transfer in wastewater bacteria that degrade metformin.…”