Functional characterization of the Υ-aminobutyric acid transporter from mycobacterium smegmatis MC2 155 reveals sodium-driven GABA transport

Ana Pavić, Yurui Ji, Agnese Serafini, Acely Garza-Garcia, Martin J. McPhillie, Alexandra O. M. Holmes, Luiz Pedro Sorio de Carvalho, Yingying Wang, Mark Bartlam (Lead / Corresponding author), Adrian Goldman (Lead / Corresponding author), Vincent L. G. Postis (Lead / Corresponding author)

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Abstract

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 g-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SECMALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported g-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of b-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that g-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that g-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 g-aminobutyric acid transport protein from mycobacteria.

Original languageEnglish
Article numbere00642-20
Number of pages14
JournalJournal of Bacteriology
Volume203
Issue number4
Early online date7 Dec 2020
DOIs
Publication statusPublished - 25 Jan 2021

Keywords

  • GABA
  • Membrane biology
  • Mycobacteria
  • Transporter

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