| Abstract
| - The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linkedglycosyl donor (Dol-PP-GlcNAc2Man9Glc3), which is assembled by the series of membrane-boundglycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conservedthroughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabledidentification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two ofthe mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymesappear to catalyze only two of the remaining four unannotated transformations. To address this issue, abiochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defineddolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia colioverexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out anα1,3-mannosylation, followed by an α1,6-mannosylation, to form the first branched pentasaccharideintermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis ofthe dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function ofAlg11, which is to catalyze the next two sequential α1,2-mannosylations. The elucidation of the dualfunction of each of these enzymes thus completes the identification of the entire ensemble ofglycosyltransferases that comprise the dolichol pathway.
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