Information on EC 2.4.1.229 - [Skp1-protein]-hydroxyproline N-acetylglucosaminyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.4.1.229
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RECOMMENDED NAME
GeneOntology No.
[Skp1-protein]-hydroxyproline N-acetylglucosaminyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-N-acetyl-alpha-D-glucosamine + [Skp1-protein]-trans-4-hydroxy-L-proline = UDP + [Skp1-protein]-O-(N-acetyl-alpha-D-glucosaminyl)-trans-4-hydroxy-L-proline
show the reaction diagram
; Requires dithiothreitol and a divalent cation for activity. This enzyme commences the building up of a pentasaccharide (Galalpha1-6Galalpha1-L-Fucalpha1-2Galbeta1-3GlcNAc) on Hyp-143 of the Dictyostelium protein Skp1, which is required for the ubiquitination of cell-cycle regulatory proteins and transcription factors. The fucose residue is probably in the alpha configuration. The specificity of the enzyme for Skp1-Hyp-143 and its high affinity for this substrate suggests that it is the GlcNAc-transferase that modifies Skp1 in vivo
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
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SYSTEMATIC NAME
IUBMB Comments
UDP-N-acetyl-alpha-D-glucosamine:[Skp1-protein]-trans-4-hydroxy-L-proline N-acetyl-alpha-D-glucosaminyl-transferase
Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium Skp1 is modified by the linear pentasaccharide Galalpha1-6Galalpha1-L-Fucalpha1-2Galbeta1-3GlcNAc, which is attached to a hydroxyproline residue at position 143. This enzyme catalyses the first step in the building up of the pentasaccharide by attaching an N-acetylglucosaminyl group to the hydroxyproline residue. It requires dithiothreitol and a divalent cation for activity.
CAS REGISTRY NUMBER
COMMENTARY hide
256531-81-4
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37277-59-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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
UDP-N-acetylglucosamine + hydroxyproline-peptide
UDP + (N-acetyl-D-glucosaminyl)hydroxyproline peptide
show the reaction diagram
UDP-N-acetylglucosamine + polypeptide-hydroxyamino acid
UDP + polypeptide-O-(alpha-N-acetyl-D-glucosaminyl)hydroxyamino acid
show the reaction diagram
UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline
UDP + [Skp1-protein]-O-(alpha-N-acetyl-D-glucosaminyl)hydroxyproline
show the reaction diagram
UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline
UDP + [Skp1-protein]-O-(N-acetyl-D-glucosaminyl)hydroxyproline
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-acetylglucosamine + polypeptide-hydroxyamino acid
UDP + polypeptide-O-(alpha-N-acetyl-D-glucosaminyl)hydroxyamino acid
show the reaction diagram
UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline
UDP + [Skp1-protein]-O-(alpha-N-acetyl-D-glucosaminyl)hydroxyproline
show the reaction diagram
Q8MZN0
enzyme is involved in O-glycosylation of Skp1-protein and mucin formation
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UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline
UDP + [Skp1-protein]-O-(N-acetyl-D-glucosaminyl)hydroxyproline
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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activates, best at 10 mM
Mn2+
absolutely dependent on
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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weak inhibition
CTP
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weak inhibition
EDTA
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complete inhibition at 5 mM
Na+
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inhibitory at concentrations above 0.05 M
UMP
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best inhibitor
additional information
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Skp-1-protein isoform containing unhydroxylated proline143 is probably inhibitory
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Brij-35
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slightly stimulating
dithiothreitol
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activates, best at 5 mM
DTT
absolutely dependent on
NP-40
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slightly stimulating
Triton X-100
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slightly stimulating
Tween 20
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stimulates, less efficient than Tween 80
Tween 80
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stimulates
Uracil
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stimulates the purified enzyme at 0.1 mM
additional information
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requires reducing condition
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6
hydroxyproline-peptide
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pH 7.8, 30°C
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0.00056
Skp-1-protein
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pH 7.8, 30°C
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0.00016
UDP-GlcNAc
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pH 7.8, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00023
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purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
neutral pH-optimum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 29600, mutant GnT51(1-282), SDS-PAGE; x * 44000, mutant GnT51(1-369+), SDS-PAGE; x * 47500, wild-type enzyme and mutants K23R, D102A, H104D, and L276S, SDS-PAGE
monomer
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1 * 51000, SDS-PAGE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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partially purified enzyme, unstable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Tween 20 and Tween 80 stabilize
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
130000fold
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recombinant His-tagged hybrid Gnt1, formed of Dictyostelium discoideum Gnt1/Dictyostelium purpureum Gnt1, from Escherichia coli by nickel affinity chromatography
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recombinant wild-type enzyme and mutants from Escherichia coli as CBD-intein tagged proteins, to near homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of a hybrid Gnt1, the coding sequence is generated from the 16 amino acid exon 1 of Dictyostelium discoideum Gnt1 and exon 2 of Dictyostelium purpureum Gnt1, and expression in Escherichia coli as His-tagged protein
gene GnT51, DNA sequence determination and analysis, functional expression of wild-type and mutants as fusion proteins with CBD-intein, in Escherichia coli strain ER2566, autocleavage of tag moiety
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D102A
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site-directed mutagenesis, no remaining activity
H104D
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site-directed mutagenesis, no remaining activity
K23R
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site-directed mutagenesis, unaltered enzyme activity
L276S
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site-directed mutagenesis, 7% remaining activity compared to the wild-type enzyme
additional information
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C-terminal deletion mutants GnT51(1-282) and GnT51(1-369+) show no remaining activity