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The Biochemical Characterization of VC0430: A Glutamate and Glutamine Binding SBP from the Poorly Understood Secondary Active Transporter TAXI-TRAP Family Found in Vibrio cholerae

Davies, Joseph (2023) The Biochemical Characterization of VC0430: A Glutamate and Glutamine Binding SBP from the Poorly Understood Secondary Active Transporter TAXI-TRAP Family Found in Vibrio cholerae. Master of Science by Research (MScRes) thesis, University of Kent,. (doi:10.22024/UniKent/01.02.101775) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided) (KAR id:101775)

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https://doi.org/10.22024/UniKent/01.02.101775

Abstract

The import of solutes into the bacterial cytoplasm involves many different membrane transporter types with some being active transport systems. Of these active transporters exist a group of secondary active transporters that require an SBP for substrate binding known as TAXI-TRAP transporters. To date, no biochemical characterisation of a TAXI-TRAP SBP exists in the literature. In Vibrio cholerae, VC0430 is an SBP from a TAXI-TRAP transporter that binds glutamate and glutamine in vitro. In this study we biochemically characterize the TAXI-TRAP SBP VC0430 by determining the specificity and Kd for L-glutamate, D-glutamate, and L-glutamine by intrinsic tryptophan fluorescence. We then generated a homology model of VC0430 and mapped it against the only known structure of a TAXI-TRAP SBP, the glutamate/glutamine binding protein TTGluBp. From this structural alignment we were able to identify key binding site residues involved in substrate binding and mutated them to alanine to determine the key substrate selectivity determinants of VC0430. Furthermore, we also optimized crystallography conditions of VC0430 in the substrate bound and apo states for structural determination by X-ray diffraction. We revealed that substrate binding in VC0430 is heavily dependent on specific hydrogen bonds with the amine group of glutamate/glutamine and specific non-polar interactions with two aromatic residues in the binding site. We further speculated on the role of glutamate/glutamine in the survival of the bacteria in its environment. Particularly in the role of glutamate is a precursor for a compatible solute that is required for survival in a high saline environment, highlighting VC0430 as a potential drug target.

Item Type: Thesis (Master of Science by Research (MScRes))
Thesis advisor: Mulligan, Chris
DOI/Identification number: 10.22024/UniKent/01.02.101775
Uncontrolled keywords: Biochemistry, Transporter, Binding, Protein
Subjects: Q Science > QD Chemistry > QD431 Organic Chemistry- Biochemistry- Proteins, peptides, amino acids
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: University of Kent (https://ror.org/00xkeyj56)
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 20 Jun 2023 17:10 UTC
Last Modified: 21 Jun 2023 15:53 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/101775 (The current URI for this page, for reference purposes)

University of Kent Author Information

Davies, Joseph.

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