Literature summary for 2.4.1.232 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.

Search for more literature for 2.4.1.232

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
D280N	site-directed mutagenesis, inactive mutant	Saccharomyces cerevisiae
H389A	site-directed mutagenesis, inactive mutant	Saccharomyces cerevisiae
additional information	construction of a yeast DELTAMNN9 enzyme deletion strain, presence of inactive full-length ScMnn9 protein partially rescues the DELTAMNN9 phenotype	Saccharomyces cerevisiae
R209A	site-directed mutagenesis, inactive mutant	Saccharomyces cerevisiae

KM Value [mM]

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

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
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

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

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	P39107	-	-
Saccharomyces cerevisiae ATCC 204508	P39107	-	-

Purification (Commentary)

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-methylumbelliferyl-alpha-D-Man + GDP-mannose	-	Saccharomyces cerevisiae	4-methylumbelliferyl-alpha-D-Man-(1->6)-D-Man + GDP	-	?
4-methylumbelliferyl-alpha-D-Man + GDP-mannose	-	Saccharomyces cerevisiae ATCC 204508	4-methylumbelliferyl-alpha-D-Man-(1->6)-D-Man + GDP	-	?
GDP-mannose + 6-O-alpha-D-mannopyranosyl-D-mannopyranose	-	Saccharomyces cerevisiae	GDP + alpha-(1->6)-D-mannotriose	-	?
GDP-mannose + 6-O-alpha-D-mannopyranosyl-D-mannopyranose	-	Saccharomyces cerevisiae ATCC 204508	GDP + alpha-(1->6)-D-mannotriose	-	?
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	?	-	?
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

Synonyms	Comment	Organism
Mnn9	-	Saccharomyces cerevisiae
ScMnn9p	-	Saccharomyces cerevisiae

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
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
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
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
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