Information on EC 2.4.1.221 - peptide-O-fucosyltransferase

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The expected taxonomic range for this enzyme is: Bilateria

EC NUMBER
COMMENTARY hide
2.4.1.221
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RECOMMENDED NAME
GeneOntology No.
peptide-O-fucosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
transfers an alpha-L-fucosyl residue from GDP-beta-L-fucose to the serine hydroxy group of a protein acceptor
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycosyl group transfer
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-
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transglycosylation
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Other types of O-glycan biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
GDP-beta-L-fucose:polypeptide O-alpha-L-fucosyltransferase
Involved in the biosynthesis of O-fucosylated epidermal growth factor (EGF) and thrombospondin type 1 repeats. The attachment of O-linked fucose to serine or threonine occurs on EGF domains within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys.
CAS REGISTRY NUMBER
COMMENTARY hide
9033-08-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Candida elegans
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Manually annotated by BRENDA team
rat
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
GDP-6-alkynyl fucose + protein
?
show the reaction diagram
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6-alkynyl fucose is efficiently incorporated onto EGF repeats, TSRs, and N-glycan on Lfng and N-glycans on a number of proteins in crude lysates of CHO cells, e.g. the O-fucosylation site in EGF3 of mouse Notch1 and elongated by Lfng, mass spectrometry analysis, overview. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or click reaction, azido-biotin allows tagging and detection of 6AF-modified proteins
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?
GDP-Fuc + biotin-DHPCTQALGNPCLNGGSCVPREATYECLCPGGFSGLHCEKG
?
show the reaction diagram
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peptide of the fourth EGF domain of agrin (EGF4) is used as an acceptor substrate with biotin conjugated at the N-terminus
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-
?
GDP-fucose + TSR1
GDP + fucosyl-TSR1
show the reaction diagram
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-
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-
?
GDP-fucose + TSR2
GDP + fucosyl-TSR2
show the reaction diagram
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-
-
-
?
GDP-fucose + TSR3
GDP + fucosyl-TSR3
show the reaction diagram
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-
-
-
?
GDP-fucose + TSR4
GDP + fucosyl-TSR4
show the reaction diagram
GDP-L-fucose + Notch
?
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
GDP-fucose + TSR4
GDP + fucosyl-TSR4
show the reaction diagram
Q9Y2G5
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
activates
additional information
Mg2+ is not required for catalytic activity by POFUT1, glycosyltransferases adopting GT-B folds are metal-independent
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
factor VII EGF-1
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inhibition above 0.015 mM
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Zn2+
complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006 - 0.015
factor VII EGF
0.004 - 0.0098
GDP-fucose
0.0295
TSR4
pH and temperature not specified in the publication
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additional information
additional information
steady-state kinetic measurements of wild-type and mutant POFUT2
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.4
GDP-fucose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
gene is widely expressed in human tissues
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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OFUT1 promotes transcytosis of Notch from the apical plasma membrane to the adherens junctions
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
44000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
the N- and the C-terminal domains adopt Rossmann-like folds, which are formed by a central beta-sheet surrounded by alpha-helices on both sides and these constitute the typical signature of a GT-B fold. The donor sugar, GDP-fucose, is localised in the interface where the two domains face each other
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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glycoprotein with more than one high mannose type oligosaccharide chain
additional information
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contains a KDEL-like sequence at the C-terminus for retention in the endoplasmic reticulum
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant truncated enzyme in apoform or complex with GDP-fucose, or GDP, or GDP and Mn2+, sitting drop method, 0.001 ml of 30 mg/ml protein is mixed with 0.001 ml of precipitant solution containing 100 mM HEPES, 100 mM MgCl2, 20% PEG 3350, pH 7.5, with or without 5 mM GDP, or 100 mM HEPES, 2% PEG 400, 1.8 M ammonium sulfate, pH 6.5, or 100 mM BIS-TRIS, 2 M ammonium sulfate, pH 6.0, 18°C, two crystals forms, X-ray diffraction structure determination and analysis at 1.54-2.60 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 55
dependent on ligands present, Mn2+ shifts the optimum to 50°C, GDP to 55°C, overview
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant truncated POFUT1_26-382 from Pichia pastoris by affinity and ion exchange chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
embryonic stem cells transfected with wild-type or mutant R245A
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expression of a truncated form of CePOFUT1, comprising amino acids 26-382, excluding the signal sequence and the retention endoplasmic reticulum localisation sequence, as a secreted protein in Pichia pastoris
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D242A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows about 10% increased activity compared to the wild-type enzyme
D244A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
D309N
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F199A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F261A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
F357A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
N43A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows highly reduced activity compared to the wild-type enzyme
R240A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R240K
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant
R40A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
W245A
site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, the mutant shows reduced activity compared to the wild-type enzyme
R254A
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expression of a mutant, Ofut1R245A, lacking fucosyltransferase activity, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development are evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose
R275A
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is catalytically dead. Ofut1 and mutant both bind the Notch extracellular domain, and expression of the mutant in cultured cells can increase both the amount and the ligand-binding activity of secreted Notch extracellular domain. Complete loss of ofut1 activity results in a strong phenotype mimicking a loss of Notch activity that is rescued to larval viability by the expression of mutant R275A, it rescues Notch receptor activity in these ofut1 mutant cells and leads to phenotypes mimicking a loss of fringe activity
D297A
site-directed mutagenesis, the mutant shows 15% activity compared to the wild-type enzyme
E396A
site-directed mutagenesis, the mutant shows 8% activity compared to the wild-type enzyme
E54A
site-directed mutagenesis, inactive mutant
R294A
site-directed mutagenesis, inactive mutant
W273a
site-directed mutagenesis, W273 is involved in controlling movements of the N- and C-terminal domain relative to each other during the catalytic cycle, and 90% activity is lost in mutant W273A
R245A
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is fucosylation-defective, has a dominant negative effect on wild-type Pofut1, since Pofut1 activity in Pofut1-/- cells expressing Pofut1 R245A is markedly reduced in the in vitro assay
S1726A
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mutant protein shows no activity, loss of O-fucosylation causes a gain of function for muscle agrin such that it stimulates acetylcholine receptors, clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology