Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis |
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Authors: | Alexander Striebeck David A Robinson Alexander W Schüttelkopf Daan M F van Aalten |
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Institution: | 1.Division of Molecular Microbiology, University of Dundee, Dundee DD1 5EH, UK;2.Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee DD1 5EH, UK;3.MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK |
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Abstract: | The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of ScMnn9 in complex with GDP and Mn2+, defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, ScMnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for ScMnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the ScMnn9 protein and its product, but not ScMnn9 catalytic activity, are required to activate subsequent ScVan1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process. |
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Keywords: | cell wall glycobiology glycosyltransferase mannan M-Pol I protein crystallography |
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