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

for references in articles please use BRENDA:EC2.4.1.229
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The enzyme appears in viruses and cellular organisms

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EC NUMBER
COMMENTARY hide
2.4.1.229
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RECOMMENDED NAME
GeneOntology No.
[Skp1-protein]-hydroxyproline N-acetylglucosaminyltransferase
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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.
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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
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
additional information
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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
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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activates, best at 10 mM
Mn2+
absolutely dependent on
additional information
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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
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00023
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purified enzyme
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
neutral pH-optimum
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29600
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x * 29600, mutant GnT51(1-282), SDS-PAGE
44000
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x * 44000, mutant GnT51(1-369+), SDS-PAGE
45000
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gel filtration
47500
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x * 47500, wild-type enzyme and mutants K23R, D102A, H104D, and L276S, SDS-PAGE
51000
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1 * 51000, SDS-PAGE
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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
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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partially purified enzyme, unstable
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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
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LINKS TO OTHER DATABASES (specific for EC-Number 2.4.1.229)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)