Information on EC 3.2.1.183 - UDP-N-acetylglucosamine 2-epimerase (hydrolysing)

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

EC NUMBER
COMMENTARY hide
3.2.1.183
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RECOMMENDED NAME
GeneOntology No.
UDP-N-acetylglucosamine 2-epimerase (hydrolysing)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-N-acetyl-alpha-D-glucosamine + H2O = N-acetyl-D-mannosamine + UDP
show the reaction diagram
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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CMP-N-acetylneuraminate biosynthesis II (bacteria)
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SYSTEMATIC NAME
IUBMB Comments
UDP-N-acetyl-alpha-D-glucosamine hydrolase (2-epimerising)
The enzyme is found in mammalian liver, as well as in some pathogenic bacteria including Neisseria meningitidis and Staphylococcus aureus. It catalyses the first step of sialic acid (N-acetylneuraminic acid) biosynthesis. The initial product formed is the alpha anomer, which rapidly mutarotates to a mixture of anomers [2]. The mammalian enzyme is bifunctional and also catalyses EC 2.7.1.60, N-acetylmannosamine kinase. cf. EC 5.1.3.14, UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cf. EC5.1.3.14, bifunctional
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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-N-acetyl-alpha-D-glucosamine + H2O
?
show the reaction diagram
UDP-N-acetyl-alpha-D-glucosamine + H2O
N-acetyl-alpha-D-mannosamine + UDP
show the reaction diagram
UDP-N-acetyl-alpha-D-glucosamine + H2O
N-acetyl-D-mannosamine + UDP
show the reaction diagram
UDP-N-alpha-D-acetylglucosamine + H2O
N-acetyl-alpha-D-mannosamine + UDP
show the reaction diagram
additional information
?
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bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase consisting of an N-terminal epimerase and a C-terminal kinase domain. The epimerase domain converts UDP-GlcNAc to ManNAc and the kinase domain phosphorylates ManNAc to ManNAc-P
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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-alpha-D-glucosamine + H2O
N-acetyl-D-mannosamine + UDP
show the reaction diagram
additional information
?
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bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase consisting of an N-terminal epimerase and a C-terminal kinase domain. The epimerase domain converts UDP-GlcNAc to ManNAc and the kinase domain phosphorylates ManNAc to ManNAc-P
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CMP-N-acetylneuraminic acid
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CMP-Neu5Ac
feedback-inhibits the UDPGlcNAc 2-epimerase activity of GNE by binding to its allosteric site
CMP-sialic acid
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feedback inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026 - 11
UDP-N-acetyl-alpha-D-glucosamine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.33 - 4.7
UDP-N-acetyl-alpha-D-glucosamine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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SDS-PAGE
47000
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SDS-PAGE
75000
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monomer, SDS-PAGE, bifunctional enzymatic activity
79000
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calculated from cDNA
80000
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x * 80000, recombinant wild-type and mutant enzymes, SDS-PAGE
450000
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gel filtration, dimer. Dimer does not show UDP-GlcNAc 2-epimerase activity. Incubation of the dimer with UDP-N-acetylglucosamine leads to reassembly of the fully active hexamer; gel filtration, hexamer, bifunctional enzyme complex
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 80000, recombinant wild-type and mutant enzymes, SDS-PAGE
hexamer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of of the selenomethionine-substituted UDP-GlcNAc 2-epimerase enzyme from Escherichia coli is determined at 2.5 resolution by multiple-wavelength anomalous dispersion (MAD) phasing. A structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase is shown
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, limited stability upon storage at 4 °C. The enzymatic activity is lost upon centrifugal concentration using ultrafiltration
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
neuC gene is cloned into an intein expression vector
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purification from Escherichia coli results in 100 microgram/l pure and structurally intact protein. For insect cells, purification methods are established which result in up to 50 mg/l pure, soluble, and active protein
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using a single ion exchange chromatographic step. The resulting protein is estimated to be 80% pure
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using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as a His-tagged fusion protein
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expressed as a His-tagged fusion protein in Escherichia coli
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expression of recombinant wild-type and mutant enzymes
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functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In Escherichia coli, up to 2mg protein/l cell culture is expressed, in yeast cells 0.4mg/l, up to 100 mg/l, are expressed in insect cells. In all three cell systems, insoluble protein aggregates are also observed
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gene GNE
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gene Gne1, sequence comparison with the human isozyme Gne1; gene Gne2, sequence comparison with the human isozyme Gne2
recombinant bifunctional enzyme is expressed in COS7 cells
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UDP-GlcNAc 2-epimerase/ManNAc kinase from rat is overexpressed in Spodoptera frugiperda cells (Sf9 cells) using a baculovirus expression system
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
treatment of knock-in Gne p.M712T mouse model with N-acetylmannosamine, the Gne transcript expression, in particular mGne2, increases significantly, likely resulting in increased Gne enzymatic activities, especially in kidney and skeletal muscle; treatment of knock-in Gne p.M712T mouse model with N-acetylmannosamine, the Gne transcript expression, in particular mGne2, increases significantly, likely resulting in increased Gne enzymatic activities, especiallyin kidney and skeletal muscle
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D176V
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naturally occuring mutation involved in hereditary inclusion body myopathy, the mutant enzyme shows 83% reduced UDP-N-acetyl alpha-D-glucosamine epimerase activity compared to the wild-type, N-acetyl mannosamine kinase activity is also reduced but to a lesser extent
V572L
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naturally occuring mutation involved in hereditary inclusion body myopathy, the mutant enzyme shows 44% reduced UDP-N-acetyl alpha-D-glucosamine epimerase activity compared to the wild-type, N-acetyl mannosamine kinase activity is laos reduced to a higher extent
D100N
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mutant is found to be significantly catalytically compromised (kcat reduced by 1000)
D131N
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mutant is found to be significantly catalytically compromised (kcat reduced by 1000), 2-acetamidoglucal is released from the active site during catalysis, providing direct evidence that the enzyme is capable of catalyzing the anti elimination of UDP from UDP-GlcNAc
E122Q
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mutant is found to be significantly catalytically compromised (kcat reduced by 1000), 2-acetamidoglucal is released from the active site during catalysis, providing direct evidence that the enzyme is capable of catalyzing the anti elimination of UDP from UDP-GlcNAc
D100N
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mutant is found to be significantly catalytically compromised (kcat reduced by 1000)
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E122Q
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mutant is found to be significantly catalytically compromised (kcat reduced by 1000), 2-acetamidoglucal is released from the active site during catalysis, providing direct evidence that the enzyme is capable of catalyzing the anti elimination of UDP from UDP-GlcNAc
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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a GNE-deficient HEK293 cell line proves its potential for the production of glycoproteins with modified sialylation, gaining therapeutic and diagnostic glycoproteins, and efficient application of metabolic oligosaccharide engineering