title: Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.
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 Mn(2+), 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.
authors.affiliation: Division of Molecular Microbiology, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Striebeck
authors.forename: David A
authors.initials: DA
authors.lastname: Robinson
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Schüttelkopf
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000378:metabolism; D008351:Mannans / Q000378:metabolism*; D008364:Mannosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C086605:MNN9 protein, S cerevisiae; D008351:Mannans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C063125:VAN1 protein, S cerevisiae; D006153:Guanosine Diphosphate; D008364:Mannosyltransferases
keywords: M-Pol I; cell wall; glycobiology; glycosyltransferase; mannan; protein crystallography
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