Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine- organocation superfamily, a potential γ-aminobutyric acid transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity and Size Exclusion Chromatography-Multi Angle Laser Light Scattering analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid in vitro and when over-expressed in E. coli. Additionally, transport was greatly reduced in the presence of β-alanine, suggesting that it could be either substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.