Bacterial cell envelope glycans are a valuable resource industrially and pharmacologically. Their production enables bacteria to withstand harsh environmental stresses including protection against antimicrobial agents, can be crucial in the formation of biofilms and, can be catastrophic to the host, causing an overwhelming inflammatory response that can ultimately lead to sepsis. Synthesis of these glycostructures is initiated by one of two enzyme families: the polyisoprenyl-phosphate N- acetylamino sugar-1-phosphate transferases (PNPTs) or the polyisoprenyl-phosphate hexose-1- phosphate transferases (PHPTs) that transfer the initial sugar-1-phosphate from a nucleotide-sugar precursor onto a universal polyisoprenoid lipid carrier. Unlike PNPT, PHPTs are unique to prokaryotes and are priming enzymes in the synthesis of surface polysaccharides of varying complexity. WcaJ is a PHPT protein that initiates the synthesis of colanic acid in the Enterobacteriaceae. Colanic acid is a high molecular weight capsular polysaccharide that protects against environmental stresses such as low temperature conditions, resistance to desiccation and antimicrobial compounds. In this study, WcaJ was utilised as a model PHPT member to investigate the structure and functional mechanisms of this family of proteins. The topology of WcaJ experimentally validated identifying a novel topological arrangement whereby the C-terminal tail and the soluble loop domain both reside in the cytoplasm. This arrangement is the result of ‘horseshoe’ structure formed by the terminal transmembrane helix; a model that is proposed extends to all PHPT members. It was further identified that WcaJ forms homoologomers mediated by self-interaction of this helix and that conserved charged residues may play a role in this interaction. Finally, in silico modelling of the cytoplasmic loop domain suggests that it is structurally analogous to the Rossmann fold nucleotide binding domains of UDP-sugar dehydrogenases. Biochemical analysis of WcaJ and other PHPT proteins suggests that the cytoplasmic loop domain could provide a site for NAD reduction that could allow PHPT to perform dual enzymatic functions. It is proposed that this could provide a mechanism for self-regulation and/or regulation of the biosynthetic pathway. Together, the data gathered within this study have provided insights into the structural and functional complexity of this family of proteins that will be a valuable resource for future study.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726344 |
| Date | January 2017 |
| Creators | Ford, Amy |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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