title: Structure-based mutagenic analysis of mechanism and substrate specificity in mammalian glycosyltransferases: porcine ST3Gal-I.
abstract: Sialyltransferases (STs) play essential roles in signaling and in the cellular recognition processes of mammalian cells by selectively installing cell-surface sialic acids in an appropriate manner both temporally and organ-specifically. The availability of the first three-dimensional structure of a mammalian (GT29) sialyltransferase has, for the first time, allowed quantitative structure/function analyses to be performed, thereby providing reliable insights into the roles of key active site amino acids. Kinetic analyses of mutants of ST3Gal-I, in conjunction with structural studies, have confirmed the mechanistic roles of His302 and His319 as general acid and base catalysts, respectively, and have quantitated other interactions with the cytosine monophosphate-N-acetyl β-neuraminic acid donor substrate. The contributions of side chains that provide key interactions with the acceptor substrate, defining its specificity, have also been quantitated. Particularly important transition-state interactions of 2.5 and 2.7 kcal mol(-1) are found between the acceptor axial 4-hydroxyl and the conserved side chains of Gln108 and Tyr269, respectively. These results provide a basis for the engineering of mammalian STs to accommodate non-natural substrate analogs that should prove valuable as chemical biological probes of sialyltransferase function.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Bojana
authors.initials: B
authors.lastname: Rakic
authors.forename: Francesco V
authors.initials: FV
authors.lastname: Rao
authors.forename: Karen
authors.initials: K
authors.lastname: Freimann
authors.forename: Warren
authors.initials: W
authors.lastname: Wakarchuk
authors.forename: Natalie C J
authors.initials: NC
authors.lastname: Strynadka
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase