Degree
Doctor of Philosophy
Program
Microbiology and Immunology
Supervisor
Dr. Miguel Valvano
Abstract
Enzymes of the polyisoprenyl-phosphate hexose-1-phosphate transferase (PHPT) family are integral membrane proteins that initiate the synthesis of glycans by catalyzing the transfer of a hexose-1-phosphate sugar from UDP-hexose to the lipid carrier undecaprenyl phosphate (Und-P). These glycans such as O antigen and exopolysaccharide (EPS) provide bacteria with protection and adaptation to the environment and host immune factors. The role of PHPT proteins in initiation and the absence of any eukaryotic homologues make them an attractive target for novel antimicrobials; however study of these proteins is difficult due to the presence of multiple transmembrane helices. A requirement of the C-terminal domain for catalytic activity has been demonstrated in vitro, but the importance of specific regions and/or residues for the activity of these enzymes was not understood and was investigated in this work. The galactose-1-phosphate transferase WbaP of S. enterica initiates the synthesis of O antigen and was used as a model to study PHPT proteins. In vivo and in vitro functional assays of WbaP allowed us to show that a soluble loop region affects O antigen chain length distribution and the C-terminal domain (WbaPCT) containing one putative transmembrane (TM) domain and tail region is sufficient for transferase activity in vivo. The location of the WbaP tail region was found to be cytoplasmic and to contain highly conserved residues essential for activity. In this work a thioredoxin (TrxA) fusion was used as a tool to improve protein folding, which allowed for the solubilization and purification of WbaPCT. Obtaining a pure WbaPCT sample has allowed for the biochemical characterization of this protein revealing that this region is sufficient to maintain specificity for undecaprenyl phosphate (Und-P). Characterization of two glucose-1-phosphate transferases from Escherichia coli K-12 and Caulobacter crescentus revealed that some members of the PHPT family are able to utilize a second sugar substrate leading to the synthesis of a different glycan. These studies will pave the way to a detailed structural and mechanistic understanding of these proteins.
Recommended Citation
Patel, Kinnari B., "Structure-Function Analysis of Enzymes of the Polyisoprenyl-Phosphate Hexose-1-Phosphate Transferase family" (2011). Electronic Thesis and Dissertation Repository. 269.
https://ir.lib.uwo.ca/etd/269