Substrate specificity of the dolichol phosphate mannose: glucosaminyl phosphatidylinositol alpha1-4-mannosyltransferase of the glycosylphosphatidylinositol biosynthetic pathway of African trypanosomes.
The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN alpha 1-6-D-myo-inositol-1-PO4-sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI alpha 1-4-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-GlcN alpha 1-6-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-PI alpha 1-4-mannosyltransferase.
Smith
T K
TK
Department of Biochemistry, University of Dundee, Scotland.
Cottaz
S
S
Brimacombe
J S
JS
Ferguson
M A
MA
D000818:Animals; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004288:Dolichol Phosphates / Q000737:chemistry / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008360:Mannosephosphates / Q000737:chemistry / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000378:metabolism*
D004187:Disaccharides; D004288:Dolichol Phosphates; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008360:Mannosephosphates; D009844:Oligosaccharides; D008364:Mannosyltransferases; C066968:dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichol alpha-1,3-mannosyltransferase