Information on EC 2.7.1.60 - N-acylmannosamine kinase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
2.7.1.60
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RECOMMENDED NAME
GeneOntology No.
N-acylmannosamine kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + N-acyl-D-mannosamine = ADP + N-acyl-D-mannosamine 6-phosphate
show the reaction diagram
acts on the acetyl and glycolyl derivatives
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
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CMP-N-acetylneuraminate biosynthesis I (eukaryotes)
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Metabolic pathways
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N-acetylneuraminate and N-acetylmannosamine degradation I
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metabolism of amino sugars and derivatives
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SYSTEMATIC NAME
IUBMB Comments
ATP:N-acyl-D-mannosamine 6-phosphotransferase
Acts on the acetyl and glycolyl derivatives.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-53-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain B
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-
Manually annotated by BRENDA team
NMRI
Uniprot
Manually annotated by BRENDA team
Sprague-Dawley
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + N-acetyl-D-mannosamine
ADP + N-acetyl-D-mannosamine 6-phosphate
show the reaction diagram
ATP + N-acetyl-D-mannosamine
ADP + N-acetyl-D-mannosamine-6-phosphate
show the reaction diagram
ATP + N-acyl-D-mannosamine
ADP + N-acyl-D-mannosamine 6-phosphate
show the reaction diagram
ATP + N-glycolylmannosamine
N-glycolylmannosamine-6-phosphate
show the reaction diagram
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-
-
-
?
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
ATP + N-acetyl-D-mannosamine
ADP + N-acetyl-D-mannosamine 6-phosphate
show the reaction diagram
ATP + N-acyl-D-mannosamine
ADP + N-acyl-D-mannosamine 6-phosphate
show the reaction diagram
additional information
?
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Q9Y223
the enzyme is a bifunctional enzyme uridine diphosphate 1-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, i.e. UDP-GlcNAc 2-epimerase/ManNAc kinase or GNE. The N-terminal domain carries out UDP-GlcNAc epimerase function, whereas the C-terminal domain is responsible for ManNAc kinase activity
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
-
less effective
Ni2+
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less effective
Zn2+
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contains zinc
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-O-methyl-GlcNAc
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3-O-Methyl-N-acetyl-D-glucosamine
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non-competitive inhibition
3-O-methyl-N-acetylglucosamine
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5,5'-dithiobis(2-nitrobenzoic acid)
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6-O-acetyl-N-acetylmannosamine
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arsenite/2,3-dimercaptopropanol
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iodoacetamide
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N-ethylmaleimide
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N-propanoylglucosamine
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p-chloromercuribenzoate
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2-mercaptoethanol protects from inactivation
Periodate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NaCl
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ManNAc kinase activity is increased by presence of 200 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.15 - 4.4
ATP
0.093 - 0.75
N-acetyl-D-mannosamine
0.127
N-acyl-D-mannosamine
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pH 8.1, 37C, recombinant enzyme
1.2
N-glycolylmannosamine
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pH 8.1, 37C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
3-O-Methyl-N-acetyl-D-glucosamine
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pH 7.4, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.84
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recombinant enzyme
2.5
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activity of fraction with N-glycolylmannosamine
2.6
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pH 8.1, 37C, recombinant enzyme
2.76
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activity of fraction with N-glycolylmannosamine
4
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activity of fraction with N-acetylmannosamine
10.9
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purified enzyme from Sf-900 cells, pH 8.1, 37C
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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pancreatic carcinoma cell line showing low enzyme activity
Manually annotated by BRENDA team
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pancreatic
Manually annotated by BRENDA team
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normal mucosa, non-malignant mucosa, tumour
Manually annotated by BRENDA team
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homozygous or heterozygous gene knock-out cells
Manually annotated by BRENDA team
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cultured cells
Manually annotated by BRENDA team
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in distal-myopathy-with-rimmed-vacuoles muscle
Manually annotated by BRENDA team
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pancreatic carcinoma cell line showing low enzyme activity
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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in distal-myopathy-with-rimmed-vacuoles muscle
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37300
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amino acid sequence, predicted from an open reading frame
37400
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amino acid sequence, predicted from an open reading frame
70000
Western blot analysis
80000
6 * 80000, SDS-PAGE
83500
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4 * 83500, dynamic light scattering, the tetramer likely represents the active state of the enzyme in the cell
85000
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4 * 85000, gel filtration
150000
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gel filtration, ion of the monomeric 75000 Da protein self associate as dimer
334000
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dynamic light scattering
340000
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gel filtration
450000
500000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homodimer
homohexamer
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kinase domain, crystal structure
tetramer
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4 * 83500, dynamic light scattering, the tetramer likely represents the active state of the enzyme in the cell; 4 * 85000, gel filtration
trimer
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some of the deletion mutants tends to form trimers
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
kinase domain
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ManNAc kinase in complex with its substrate ManNAc, its product ManNAc 6-phosphate, with or without ADP, vapor diffusion at 18C, mixing 0.002 ml of 15 mg/ml protein in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM ManNAc, with 0.002 ml of reservoir solution containing 0.2 M calcium acetate, 0.1 M sodiumcacodylate, pH 6.5, and 40% PEG 300, 5 days, X-ray diffraction structure determination and analysis at 1.64-2.10 A resolution. No crystals by co-crystallization of hMNK with ManNAc and ADP, ATP, AMPPCP, or AMPPNP, but ternary complexes are obtained by soaking crystals of the binary hMNK-ManNAc complex replacing stepwise their mother liquor with solutions containing 0.1 M sodium cacodylate, pH 6.5, 50% PEG 300, and 20 mM ADPor ATP or nonhydrolyzable ATP derivatives
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sitting drop vapor diffusion method, using 0.2 M ammonium chloride, 20% (w/v) polyethylene glycol 6000, 0.1 M HEPES pH 7.0
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1 - 2
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pH above 12 results in almost complete disaggregation and complete loss of activity
663299
6
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pH below 6 results in almost complete disaggregation and complete loss of activity
663299
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
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stable to heating for a short period in a water-bath
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
hexameric enzyme is completely stable with 0.1 mM UDP
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purified enzyme is unstable to ammonium sulfate precipitation or freezing and thawing
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, no loss of activity observed for several months
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0C, loses about 50% of its activity in 24 hours, stable for at least 1 week in presence of 0.01 M N-acylmannosamine
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4C, enzyme can be stored without substantial loss of activity
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4C, loses about 30% of its activity during 7 days of storage
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4C, loses about 33% of its activity during 7 days of storage
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4C, stable for several days
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
MonoQ HR 5/5 column chromatography, PD-10 column chromatography, and Superdex 200 gel filtration
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Ni-NTA column chromatography and PD-10 gel column filtration
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Q Sepharose column chromatography, phenyl Sepharose column chromatography, and Superdex 200 gel filtration
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recombinant enzymes from all species, purification from Sf-900 can yield milligram amounts of soluble, active protein
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recombinant proteins using His-tag
recombinant proteins using Myc-tag
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA cloned and sequenced, expressed in Escherichia coli HB101 or INValphaF'
cloned and functionally expressed in Escherichia coli BL21
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cloning of isozymes hGNE1-hGNE8, DNA and amino acid sequence determination and analysis, quantitative real-time PCR expression analysis and sequence comparisons
cloning of isozymes mGNE1 and mGNE2, DNA and amino acid sequence determination and analysis, quantitative real-time PCR expression analysis and sequence comparison
expressed as His-tag fusion protein in Sf9, and Sf-900 cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3)pS and HEK-293T cells
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expressed in Escherichia coli strain DH5alpha
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expressed in HeLa cells
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expressed in Sf-9 cells
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expressed in Sf9 insect cells
expressed with or without His-tag in Escherichia coli BL21 Star(DE3), Saccharomyces cerevisiae, Pichia pastoris, High five, Sf9, and Sf-900 cells, low yield in Escherichia coli and yeast, high expression of enzyme in High five, and Sf-900 cells
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expression as His-tag fusion protein in Sf9 cells
expression in 293T cell
expression in CHO cells
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expression of GFP-tagged enzyme in HEK-293 cells, the expression is efficiently blocked by sh70-shRNA, sh68 has no effect on GNE protein expression because its target sequence at the 3'-UTR is not present in the GFP-hGNE vector construct
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His-tag, kinase domain expressed in Escherichia coli BL21(D3) CodonPlus-RIL
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overexpression of active enzyme by using the baculovirus/Sf9 system
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wild type an mutated proteins expressed as Myc-tag fusion proteins in COS-7 cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
GNE up-regulation occurs predominantly in pancreatic cancer but also in other malignancies
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A524V
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30% ManNAc kinase activity of the wild type enzyme
A631T
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75% ManNAc kinase activity of the wild type enzyme
A631V
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15% ManNAc kinase activity of the wild type enzyme; 65% ManNAc kinase activity of the wild type enzyme
C303V
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60% ManNAc kinase activity of the wild type enzyme
C303X
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no ManNAc kinase activity
D378Y
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45% ManNAc kinase activity of the wild type enzyme
D517A
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site-directed mutagenesis, inactive mutant
D517N
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site-directed mutagenesis, inactive mutant
E35X
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enzyme activity is severely decreased or absent in this mutant
F528C
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35% ManNAc kinase activity of the wild type enzyme
F537I
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60% ManNAc kinase activity of the wild type enzyme
G135E
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enzyme activity is severely decreased or absent in this mutant
G576E
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15% ManNAc kinase activity of the wild type enzyme
G576R
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the mutation is associated with distal myopathy with rimmed vacuoles
G708S
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5% ManNAc kinase activity of the wild type enzyme
H132Q
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50% ManNAc kinase activity of the wild type enzyme
I200F
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75% ManNAc kinase activity of the wild type enzyme
I472T
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5% ManNAc kinase activity of the wild type enzyme
I587T
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35% ManNAc kinase activity of the wild type enzyme
M743T
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the mutation, which is associated with GNE myopathy, has a 10fold lower binding affinity to alpha-actinin 2 than the intact enzyme
N519S
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20% ManNAc kinase activity of the wild type enzyme
R11W
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40% ManNAc kinase activity of the wild type enzyme
R177C
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80% ManNAc kinase activity of the wild type enzyme
V331A
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115% ManNAc kinase activity of the wild type enzyme
D176V
oral administration of the sialic acid precursor N-acetylmannosamine rescues the muscle phenotype in the transgenic Gne p.D176V mouse and partially rescues the glomerular disease and early lethality in the knockin Gne mutant M712T mouse model
M712T
naturally occuring mutation of isozyme mGne2. Tissues of the knock-in Gne p.M712T mouse model has similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression, but the mutant shows increased activity in presence of N-acetylmannosamine compared to the wild-type enzyme. M712T mouse mutants die within 72 h of birth from severe glomerular disease
D176V
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oral administration of the sialic acid precursor N-acetylmannosamine rescues the muscle phenotype in the transgenic Gne p.D176V mouse and partially rescues the glomerular disease and early lethality in the knockin Gne mutant M712T mouse model
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M712T
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naturally occuring mutation of isozyme mGne2. Tissues of the knock-in Gne p.M712T mouse model has similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression, but the mutant shows increased activity in presence of N-acetylmannosamine compared to the wild-type enzyme. M712T mouse mutants die within 72 h of birth from severe glomerular disease
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D413K
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loss of kinase activity
D413L
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loss of kinase activity
D413N
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loss of kinase activity
DELTA1-234
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monomer molecular mass of 58000 Da, complete loss of epimerase activity, 90% loss of kinase activity
DELTA1-359
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monomer molecular mass of 45000 Da, complete loss of epimerase activity, almost complete loss of kinase activity
DELTA1-39
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monomer molecular mass of 79000 Da, complete loss of epimerase activity, 75% loss of kinase activity
DELTA383-722
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monomer molecular mass of 49000 Da, almost complete loss of epimerase activity, complete loss of kinase activity
DELTA490-722
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monomer molecular mass of 61000 Da, more than 80% loss of epimerase activity, complete loss of kinase activity
DELTA597-722
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monomer molecular mass of 72000 Da, almost complete loss of epimerase activity, and kinase activity
DELTA697-722
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monomer molecular mass of 83000 Da, 25% loss of epimerase activity, 60% loss of kinase activity
DELTA717-722
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monomer molecular mass of 85000 Da, less than 10% loss of epimerase activity, 20% loss of kinase activity
H110A
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loss of epimerase activity
H132A
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loss of epimerase activity
H155A
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loss of epimerase activity
H157A
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loss of epimerase activity
H45A
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loss of epimerase activity
R420M
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loss of kinase activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
molecular biology
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GNE-deficient cells, with dramatically increased incorporation of N-acetylmannosamine analogues into glycoproteins, can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation
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