J Biol Chem, Vol. 274, Issue 53, 37717-37722, December 31, 1999

Glycosyl Fluorides Can Function as Substrates for Nucleotide Phosphosugar-dependent Glycosyltransferases^*

Brenda Lougheed, Hoa D. Ly, Warren W. Wakarchuk^§, and Stephen G. Withers^¶

From the  Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1 and the ^§ Institute of Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada

-Galactosyl fluoride is shown to function as a substrate, in place of uridine-5'-diphosphogalactose, for the -galactosyltransferase from Neisseria meningitidis. The reaction only occurs in the presence of catalytic quantities of uridine 5'-diphosphate. In the presence of galactosyl acceptors, the expected oligosaccharide product is formed in essentially quantitative yields, reaction having been performed on multi-milligram scales. In the absence of a suitable acceptor, the enzyme synthesizes uridine-5'-diphosphogalactose, as demonstrated through a coupled assay in which uridine-5'-diphosphogalactose is converted to uridine-5'-diphosphoglucuronic acid with conversion of NAD to NADH. These glycosyl fluoride substrates therefore offer the potential of an inexpensive alternative donor substrate in the synthesis of oligosaccharides as well a means of generating steady state concentrations of nucleotide diphosphate sugars for in situ use by other enzymes. Further, they should prove valuable as mechanistic probes.