Information on EC 2.7.8.15 - UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase

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The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY hide
2.7.8.15
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RECOMMENDED NAME
GeneOntology No.
UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-N-acetyl-D-glucosamine + dolichyl phosphate = UMP + N-acetyl-D-glucosaminyl-diphosphodolichol
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
substituted phospho group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
dolichyl-diphosphooligosaccharide biosynthesis
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Metabolic pathways
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N-Glycan biosynthesis
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protein N-glycosylation (eukaryotic, high mannose)
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SYSTEMATIC NAME
IUBMB Comments
UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
70431-08-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
N402
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-
Manually annotated by BRENDA team
N402
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
X2180-1A
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
UDP-GlcNAc + dolichyl phosphate
UMP + GlcNAc-diphosphodolichol
show the reaction diagram
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initiating subsequent synthesis of Glc3Man9GlcNAc2-diphosphodolichol
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?
UDP-N-acetyl-D-glucosamine + dolichyl phosphate
?
show the reaction diagram
UDP-N-acetyl-D-glucosamine + dolichyl phosphate
UMP + N-acetyl-D-glucosaminyl-diphosphodolichol
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
UDP-N-acetyl-D-glucosamine + dolichyl phosphate
?
show the reaction diagram
UDP-N-acetyl-D-glucosamine + dolichyl phosphate
UMP + N-acetyl-D-glucosaminyl-diphosphodolichol
show the reaction diagram
additional information
?
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alg7 mutants show diminished activity of GPT, reduced steady-state levels of lipid-linked oligosaccharides and hypoglycosylated carboxypeptidase Y. Mutants also lack mitochondrial DNA including the genes that encode the components of the respiratory machinery
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Amphomycin
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Ca2+
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above 2 mM
Diumycin
dolichyl phosphate
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at high concentrations
GDP-mannose
GTP
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0.4 mM, 25-30% inhibition, enzyme from tunicamycin-resistant cells, no inhibition of wild-type enzyme up to 0.4 mM
Hg2+
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partially reversed by dithiothreitol
iodoacetamide
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Mn2+
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above 2 mM
NEM
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protected to the extent of about 50% when all of the substrates, UDP-GlcNAc, dolichyl phosphate and Mn2+ are added before addition of the sulfhydryl reagent
phosphatidylcholine
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phosphatidylethanolamine
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phosphatidylserine
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Showdomycin
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Triton X-100
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substrate protects from inhibition
tunicamycin
UDP-GlcNAc
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strong inhibition
UDP-glucose
UDP-hexanolamine
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UDP-xylose
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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0.01-0.025 mM, stimulates activity of tunicamycin-resistant cells about 20-30%
cardiolipin
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stimulates activity of enzyme in endoplasmic membrane vesicles, due to better solubilization of the dolichol phosphate
Dolichyl-phosphoryl-mannose
GDP-mannose
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stimulates by protecting the substrate UDP-GlcNAc from degradation
monogalactosyldiglyceride
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stimulates activity of enzyme in endoplasmic membrane vesicles
phosphatidylcholine
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0.001 mM/ml, 30-40% inhibition
phosphatidylethanolamine
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stimulates activity of enzyme in endoplasmic membrane vesicles
phosphatidylglycerol
phosphatidylinositol
phosphatidylserine
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solubilized enzyme is stimulated by exogenously added phospholipids in order of increasing activity: phosphatidylglycerol, phosphatidylinositol, phosphatidylserine
Phospholipid
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0007 - 0.18
dolichyl phosphate
0.0001 - 0.015
UDP-GlcNAc
additional information
additional information
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Km-value for dolichyl phosphate: 0.005 mg/0.1 ml
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 7.6
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7.4 - 7.6
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
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pH 6.5: about 60% of maximal activity, pH 8.5: about 35% of maximal activity
7 - 8.7
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pH 6.0: no activity, pH 7.0: 85% of maximal activity, pH 8.7: 64% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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with 0.5% Triton X-100 in the assay mixture
60
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with either dioleoylphosphatidylethanolamine or cardiolipin in the assay mixture
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 40
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0C: no activity, 15C: 11% of maximal activity, 25C: 73% of maximal activity, 35C: 96% of maximal activity, 40C: 57% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34000
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monomer, determined by SDS-PAGE and Western blot analysis
44700
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46000
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determined by SDS-PAGE and Western Blot analysis
46470
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theoretical
50000
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determined by SDS-PAGE and Western Blot analysis
67000
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assumed to be a dimeric form, determined by SDS-PAGE and Western blot analysis
70000
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suggested to be a biological precursor or the degraded native enzyme, determined by SDS-PAGE and Western blot analysis
330000 - 360000
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large molecular mass of the enzyme can be due to aggregation of the hydrophobic, membrane-derived enzyme rather than resulting from subunit interaction, gel filtration
additional information
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ALG7 1.5 kb mRNAs appears to be initiated at a start site downstream from that used by the 1.9 and 2.2 kb species and located bentween two in-frame ATG codons. Translation of these transcripts in vitro provides supporting evidence that the 1.5 and 1.9 kb transcripts are translationally competent, giving rise to related protein isoforms with different lengths of their NH2-terminal domains. The 1.9 kb mRNA serves in the synthesis of 36000 Da and 24000 Da species, as well as a low abundance 32000 Da protein. The 1.5 kb transcript gives rise to a translation product of 32000 Da
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 34000, the 67000 Da enzyme form is a homodimer of UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase or perhaps a hetero-oligomer with another microsomal component
additional information
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results indicate that either 70000 MW band in SDS-PAGE is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptides of 50000 Da and 46000 Da during purification
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the enzyme is not a glycoprotein of the asparagine-linked type
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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1 h, stable
35
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the enzyme from tunicamycin-resistant cells is significantly less stable than the wild-type enzyme
40
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the enzyme from tunicamycin-resistant cells is significantly less stable than the wild-type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
a 1:4 dilution leads to a 65% loss of activity
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purified enzyme is resistant to digestion with either endo-beta-N-acetylglucosaminidase H or F
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C or -70C, stable for 2 weeks
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-20C, solubilized enzyme loses 96% of its activity after 6 days, only 20% loss of activity after 6 days in presence of 0.07 mM UDP-GlcNAc
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0-4C, 20% glycerol, 0.02 mg phosphatidylglycerol/mg of protein, less than 20% loss of activity after 6 days. In absence of both glycerol and phosphatidylglycerol the enzyme is quite unstable and loses most of its activity within 24 h
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4C, completely stable for 9-10 days. No significant loss of activity after 63 days in presence of NaN3
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4C, solubilized enzyme loses most of its activity within 12 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
for expression in Saccharomyces cerevisiae
plasmid shuffling procedure in Schizosaccharomyces pombe for analyzing site-directed mutations in the cDNA of L-G1PT for their effect on enzymatic activity
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A263D
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
D252A
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mutation does not allow plasmid shuffling, indicating that the residues is essential for activity
D252A/F254I
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mutation does not allow plasmid shuffling, indicating that the residues are essential for activity
D252A/L102F
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mutation does not allow plasmid shuffling, indicating that the residues are essential for activity
DELTA395-408
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mutation fully eliminates enzyme expression in vivo
DELTA398-408
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removal of the last 11 amino acids 398-408 from the enzyme has no significant effect on the catalytic activity, thermal stability, tunicamycin binding, reticular localization, or consumption of cellular dolichol phosphate
F249A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
F257A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
F395L/S396M/I397W
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expression of the mutant enzyme
F395L/S396M/I397W/DELTA398-408
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mutation fully eliminates enzyme expression in vivo
K125A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
N182A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
N182A/I186M
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mutation does not allow plasmid shuffling, indicating that the residues are essential for activity
N182A/W122R
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mutation does not allow plasmid shuffling, indicating that the residues are essential for activity
N185A
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mutation does not allow plasmid shuffling, indicating that the residue is essential for activity
R123A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
W122R
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
Y256A
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
Y256A/F286S
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mutation allows plasmid shuffling, indicating that the residue is not essential for activity
Y170C
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mutant enzyme has almost no activity
additional information
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c.3503_3504delTC: heterozygous, two-nucleotide deletion in exon 19, causes frameshift and premature translational stop (p.L1168QfsX5), detected by PCR in all tested parents of children with mucolipidosis II
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