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Advancing mechanistic understanding of glycosyltransferases

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dc.contributor.author Gagnon, Susannah Melanie Lynn
dc.date.accessioned 2019-04-24T23:37:06Z
dc.date.available 2019-04-24T23:37:06Z
dc.date.copyright 2019 en_US
dc.date.issued 2019-04-24
dc.identifier.uri http://hdl.handle.net/1828/10754
dc.description.abstract Glycosyltransferase enzymes synthesize glycosidic linkages, generating carbohydrates and carbohydrate-linked entities ranging from cellulose, starch, and chitin to glycolipids, glycopeptides, and natural product antibiotics. These syntheses involve stereo- and regio-specific sugar transfer from an activated donor molecule, often a UDP-sugar, to an acceptor molecule. Functionally, glycosyltransferases are classified as either “retaining” or “inverting” enzymes depending on whether the stereochemical linkage of the donor substrate is conserved in the product. While inverting glycosyltransfer is mechanistically straightforward, the retaining mechanism remains poorly understood. For retaining glycosyltransferases, the central question is whether transfer occurs via a front-face “SNi-like” mechanism or through a ‘double displacement’ mechanism that invokes a glycosyl-enzyme covalent intermediate. GTA and GTB are retaining enzymes that catalyze the final step in human ABO(H) blood group A and B antigen synthesis through UDP-GalNAc or UDP-Gal transfer, respectively, to the H-antigen disaccharide acceptor. Although they have been intensively characterized, the processes of substrate recognition, mobile loop organization, and product release in GTA and GTB has long resisted explanation. Further, the question of the retaining enzyme mechanism persists, though the covalent intermediate of the proposed double displacement mechanism has been detected via mass spectrometry experiments with GTA/GTB mutants. Building on previous investigations, we have aimed to characterize and have uncovered details of mechanism, substrate binding, loop organization, and product release using a combined kinetic and structural approach. These investigations are essential not only for understanding GTA, GTB, and retaining glycosyltransferases as a whole, but also for the rational design of inhibitors. Such inhibitors could selectively target, for example, bacterial glycosyltransferases and thus would represent a new class of antimicrobials. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject glycosyltransferases en_US
dc.subject mechanism en_US
dc.subject structural biology en_US
dc.subject X-ray crystallography en_US
dc.subject human blood group enzymes en_US
dc.title Advancing mechanistic understanding of glycosyltransferases en_US
dc.type Thesis en_US
dc.contributor.supervisor Evans, S. V.
dc.degree.department Department of Biochemistry and Microbiology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.identifier.bibliographicCitation Blackler RJ, Gagnon SM, Polakowski R, Rose NL, Zheng RB, Letts JA, Johal AR, Schuman B, Borisova SN, Palcic MM, et al. 2017. Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site. Glycobiology, 27:370-380. en_US
dc.identifier.bibliographicCitation Gagnon SML, Legg MSG, Polakowski R, Letts JA, Persson M, Lin S, Zheng RB, Rempel B, Schuman B, Haji-Ghassemi O, et al. 2018. Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases. Glycobiology, 28:624-636. en_US
dc.identifier.bibliographicCitation Gagnon SML, Legg MSG, Sindhuwinata N, Letts JA, Johal AR, Schuman B, Borisova SN, Palcic MM, Peters T, Evans SV. 2017. High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release. Glycobiology, 27:966-977. en_US
dc.identifier.bibliographicCitation Gagnon SM, Meloncelli PJ, Zheng RB, Haji-Ghassemi O, Johal AR, Borisova SN, Lowary TL, Evans SV. 2015. High resolution structures of the human ABO(H) blood group enzymes in complex with donor analogs reveal that the enzymes utilize multiple donor conformations to bind substrates in a stepwise manner. J Biol Chem, 290:27040-27052. en_US
dc.description.scholarlevel Graduate en_US


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