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Literature summary extracted from

  • Striebeck, A.; Robinson, D.A.; Schuettelkopf, A.W.; van Aalten, D.M.
    Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis (2013), Open Biology, 3, 130022.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.232 recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) pLysS, expression of mutant enzymes in Saccharomyces cerevisiae strain BY4741 DELTAMNN9 Saccharomyces cerevisiae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.232 purified recombinant wild-type and mutant enzymes, sitting drop vapour diffusion, mixing of 0.0005 ml of protein solution with 0.0005 ml of preciputation solution containing 0.1 M HEPES, pH 7.5, and 2 M ammonium sulfate, crystals are soaked in 50% Na-malonate, pH 7.5, containing mersalyl acid for 16 h at 20°C, crystals of ScMnn9-D236N mutant are transferred to 50% Na-malonate, pH 7.5, and soaked with 3 mM GDP and 10 mM MnCl2 for 10 min at 20°C, X-ray diffraction structure determination and analysis at 2.0-2.2 A resolution, modeling Saccharomyces cerevisiae

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.232 D236N site-directed mutagenesis, D236 is the first aspartic acid in the canonical GT-A DxD catalytic motif, and mutation to the isosteric asparagine results in the loss of activity Saccharomyces cerevisiae
2.4.1.232 D280N site-directed mutagenesis, inactive mutant Saccharomyces cerevisiae
2.4.1.232 H389A site-directed mutagenesis, inactive mutant Saccharomyces cerevisiae
2.4.1.232 additional information construction of a yeast DELTAMNN9 enzyme deletion strain, presence of inactive full-length ScMnn9 protein partially rescues the DELTAMNN9 phenotype Saccharomyces cerevisiae
2.4.1.232 R209A site-directed mutagenesis, inactive mutant Saccharomyces cerevisiae

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.1.232 additional information
-
 additional information steady-state kinetics of wild-type ScMnn9 enzyme Saccharomyces cerevisiae

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.1.232 Golgi membrane ScMnn9p enzyme is a type II membrane proteins, possessing a short cytosolic N-terminal domain followed by a transmembrane domain that is required for anchoring to the Golgi membrane Saccharomyces cerevisiae 139
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.4.1.232 Mn2+ required, enzyme complex binding structure, overview Saccharomyces cerevisiae
2.4.1.232 additional information Ni2+, Mg2+, Ca2+, and Zn2+ cannot substitute for Mn2+ Saccharomyces cerevisiae

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.232 Saccharomyces cerevisiae P39107
-
-
2.4.1.232 Saccharomyces cerevisiae ATCC 204508 P39107
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.232 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3) pLysS by nickel affinity chromatography Saccharomyces cerevisiae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.232 4-methylumbelliferyl-alpha-D-Man + GDP-mannose
-
 Saccharomyces cerevisiae 4-methylumbelliferyl-alpha-D-Man-(1->6)-D-Man + GDP
-
 ?
2.4.1.232 4-methylumbelliferyl-alpha-D-Man + GDP-mannose
-
 Saccharomyces cerevisiae ATCC 204508 4-methylumbelliferyl-alpha-D-Man-(1->6)-D-Man + GDP
-
 ?
2.4.1.232 GDP-mannose + 6-O-alpha-D-mannopyranosyl-D-mannopyranose
-
 Saccharomyces cerevisiae GDP + alpha-(1->6)-D-mannotriose
-
 ?
2.4.1.232 GDP-mannose + 6-O-alpha-D-mannopyranosyl-D-mannopyranose
-
 Saccharomyces cerevisiae ATCC 204508 GDP + alpha-(1->6)-D-mannotriose
-
 ?
2.4.1.232 additional information both the presence and the priming activity of enzyme ScMnn9 are required for the formation of the alpha-1,6-mannose backbone of mannan proteins. Determination of the structure of the mannosyltransferase domain of ScMnn9 in complex with manganese and GDP, overview. Development of a coupled enzyme assay that involves Bacillus subtilis TN-31 aman6 (Aman6), an alpha-1,6-mannosidase, as only one additional enzyme, in contrast to the established glycosyltransferase assays where the release of GDP is measured by NADH oxidation through pyruvate kinase and lactate dehydrogenase Saccharomyces cerevisiae ?
-
 ?
2.4.1.232 additional information both the presence and the priming activity of enzyme ScMnn9 are required for the formation of the alpha-1,6-mannose backbone of mannan proteins. Determination of the structure of the mannosyltransferase domain of ScMnn9 in complex with manganese and GDP, overview. Development of a coupled enzyme assay that involves Bacillus subtilis TN-31 aman6 (Aman6), an alpha-1,6-mannosidase, as only one additional enzyme, in contrast to the established glycosyltransferase assays where the release of GDP is measured by NADH oxidation through pyruvate kinase and lactate dehydrogenase Saccharomyces cerevisiae ATCC 204508 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.4.1.232 Mnn9
-
 Saccharomyces cerevisiae
2.4.1.232 ScMnn9p
-
 Saccharomyces cerevisiae

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.232 20
-
 assay at Saccharomyces cerevisiae

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.232 7.5
-
 assay at Saccharomyces cerevisiae

General Information

EC Number General Information Comment Organism
2.4.1.232 evolution the enzyme belongs to the CAZy glycosyltransferase family GT-62 that contains metal-dependent, retaining mannosyltransferases that are predicted to possess the GT-A fold and use GDP-Man as their donor substrate Saccharomyces cerevisiae
2.4.1.232 malfunction cells expressing catalytically inactive ScMnn9p show growth kinetics comparable with those measured for cells lacking the enzyme, phenotypes of inactive enzyme mutants, overview Saccharomyces cerevisiae
2.4.1.232 additional information enzyme ScMnn9 possesses the GT-A fold and shows structural similarity to GT families 15 and 78. ScMnn9 possesses a unique extrusion that may act as a molecular ruler or multimerization domain Saccharomyces cerevisiae
2.4.1.232 physiological function ScMnn9p catalytic activity is indispensable for mannoprotein synthesis in yeast. 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. Enzyme Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis, an allosteric activator for ScVan1 polymerase activity Saccharomyces cerevisiae