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dc.contributor.authorXu, Changchangen
dc.date2010-09-23 21:14:29.363
dc.date.accessioned2010-09-24T21:28:52Z
dc.date.available2010-09-24T21:28:52Z
dc.date.issued2010-09-24T21:28:52Z
dc.identifier.urihttp://hdl.handle.net/1974/6086
dc.descriptionThesis (Master, Biochemistry) -- Queen's University, 2010-09-23 21:14:29.363en
dc.description.abstractThe O-antigenic glycan chains (O-antigen) of lipopolysaccharides (LPS) in Gram-negative bacteria are synthesized by glycosyltransferases (GTs), most of which are yet to be characterized. The LPS are important for cell surface functions in bacteria and for the host immune system, through influencing the ability of bacteria to invade tissues and to evade the immune system of the host. Shigella is a Gram-negative pathogen that causes diarrhea or dysentery in humans. The O-antigenic chain of Shigella boydii (S. boydii) serotype B14 consists of repeating units with the structure [-6-D-Galp-alpha1,4-D-GlcpA-beta1,6-D-Galp-beta1,4-D-Galp-beta1,4-D-GlcpNAc-beta1-]n. None of the genes in the S. boydii serotype B14 O-antigen gene cluster, which includes putative GT genes, have been functionally characterized. We have developed the technology to biochemically identify these novel GTs. The wfeD gene in the B14 O-antigen gene cluster was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a beta4GalT that synthesizes the Gal1-4GlcNAcalpha-R linkage. WfeD was expressed in E.coli and the activity characterized using UDP-[3H]Gal as the donor substrate and the synthetic acceptor substrate GlcNAcalpha-pyrophosphate-phenyl-undecyl (GlcNAc-PP-PhU). A His-tagged version of the enzyme was purified via Ni2+-affinity chromatography, and its disaccharide product was analyzed by liquid chromatography-mass spectrometry (LC-MS), high pressure liquid chromatography (HPLC), 1D and 2D nuclear magnetic resonance (NMR), and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn2+, Ni2+, and surprisingly also Pb2+, were able to enhance enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue within a cluster of positively charged amino acids was also found to be required for activity. This latter residue may be involved in binding the negatively charged acceptor substrate. Our study revealed that the beta4GalT WfeD is a previously unknown enzyme that has extremely low sequence similarities to any other GalT and has an unusual preference for metal ion cofactors.en
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectBiochemistryen
dc.titleCharacterization of a shigella boydii B14 galactosyltransferase involved in O-antigen synthesisen
dc.typethesisen
dc.description.degreeM.Sc.en
dc.contributor.supervisorBrockhausen, Inkaen
dc.contributor.supervisorAllingham, Johnen
dc.contributor.departmentBiochemistryen
dc.degree.grantorQueen's University at Kingstonen


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