A high-yield, enzymatic synthesis of GDP-D-[H-3]Arabinose and GDP-L-[H-3]fucose

For assays involving glycosyltransferases or transporters, several GDP-suga rs are either commercially unavailable or expensive. We describe an enzymat ic synthesis of GDP-D-[H-3]arabinose(p) and GDP-L-[H-3]fucose that yields 6 6-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min. The coupled reaction requires Mg2+, ATP, and GTP along with the ap propriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase . The latter two activities are present in a cytosolic fraction of Crithidi a fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2. Addition of commercial yeast inorganic p yrophosphatase shifts the equilibrium of the pyrophosphorylase reaction tow ard nucleotide-sugar formation. To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for gly cosyltransferases. GDP-L-[H-3]fucose functions as the donor substrate for r ecombinant human fucosyltransferase V, and GDP-D-[H-3] arabinose(p) serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes. (C) 1999 Academic Press.