Substrate specificity of the dolichol phosphate mannose: Glucosaminyl phosphatidylinositol α1-4-mannosyltransferase of the glycosylphosphatidylinositol biosynthetic pathway of African trypanosomes

Terry K. Smith, Sylvain Cottaz, John S. Brimacombe, Michael A. J. Ferguson

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    Abstract

    The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcNa1-6-D-myo-inositol-1-PO4-sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PIa1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo-inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcNa1-6-D-myo-inositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C5 and C15) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol-phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PIa1-4-mannosyltransferase.

    Original languageEnglish
    Pages (from-to)6476-6482
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume271
    Issue number11
    DOIs
    Publication statusPublished - 15 Mar 1996

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