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

• Published in: The Journal of biological chemistry Vol. 274; no. 53; pp. 37717 - 37722
• Main Authors: Lougheed, B, Ly, H D, Wakarchuk, W W, Withers, S G
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 31.12.1999
• Subjects: a-Galactosyl fluoride; a-Galactosyltransferase; Fluorides - metabolism; Glycosyltransferases - metabolism; Kinetics; Neisseria meningitidis; Recombinant Proteins - metabolism; Substrate Specificity; uridine 5'-diphosphate; uridine diphosphate galactose; Uridine Diphosphate Galactose - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.53.37717

α-Galactosyl fluoride is shown to function as a substrate, in place of uridine-5′-diphosphogalactose, for the α-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.