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Structure-based mutagenic analysis of mechanism and substrate specificity in mammalian glycosyltransferases: Porcine ST3Gal-I

TitleStructure-based mutagenic analysis of mechanism and substrate specificity in mammalian glycosyltransferases: Porcine ST3Gal-I
Publication TypeJournal Article
Year of Publication2013
AuthorsRakic, B, Rao, FV, Freimann, K, Wakarchuk, W, Strynadka, NCJ, Withers, SG
JournalGLYCOBIOLOGY
Volume23
Pagination536-545
Date PublishedMAY
ISSN0959-6658
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 beta-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.

DOI10.1093/glycob/cwt001