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Information on EC 2.4.1.221 - peptide-O-fucosyltransferase and Organism(s) Caenorhabditis elegans and UniProt Accession Q18014

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EC Tree
     2 Transferases
         2.4 Glycosyltransferases
             2.4.1 Hexosyltransferases
                2.4.1.221 peptide-O-fucosyltransferase
IUBMB Comments
The enzyme, found in animals and plants, is involved in the biosynthesis of O-fucosylated proteins. In EGF domains, the attachment of O-linked fucose to serine or threonine occurs within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys.
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This record set is specific for:
Caenorhabditis elegans
UNIPROT: Q18014
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The taxonomic range for the selected organisms is: Caenorhabditis elegans
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Reaction Schemes
hide
GDP-beta-L-fucose
+
[protein]-(L-serine/L-threonine)
=
GDP
+
[protein]-3-O-(alpha-L-fucosyl)-(L-serine/L-threonine)
+
[protein]-(L-serine/L-threonine)
=
+
[protein]-3-O-(alpha-L-fucosyl)-(L-serine/L-threonine)
Synonyms
pofut1, protein o-fucosyltransferase 1, pofut2, o-fucosyltransferase, ofut1, protein o-fucosyltransferase 2, o-fut1, o-fuct-1, alpha-6-fucosyltransferase, o-fucosyltransferase 1, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
GDP-fucose protein O-fucosyltransferase 1
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protein O-fucosyltransferase 1
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alpha-6-fucosyltransferase
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core alpha6FucT
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fucosyltransferase, guanosine diphosphofucose-glycoprotein
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GDP-fucose glycoprotein fucosyltransferase
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GDP-fucose protein O-fucosyltransferase
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GDP-fucose:polypeptide fucosyltransferase
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-
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GDP-L-fucose-glycoprotein fucosyltransferase
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GDP-L-fucose:polypeptide fucosyltransferase
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glycoprotein fucosyltransferase
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-
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guanosine diphosphofucose-glycoprotein fucosyltransferase
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N-acetyl-beta-D-glucosaminide alpha1-->6-fucosyltransferase
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N-glycan alpha-6-fucosyltransferase
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O-fucT-1
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Pofut1
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-
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POFUT2
protein O-fucosyltransferase 2
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GDP-beta-L-fucose + [protein]-(L-serine/L-threonine) = GDP + [protein]-3-O-(alpha-L-fucosyl)-(L-serine/L-threonine)
show the reaction diagram
catalytic mechanism of isozyme POFUT2 and its preference for threonine over serine residues
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 SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
GDP-beta-L-fucose:protein-(L-serine/L-threonine) O-alpha-L-fucosyltransferase (configuration-inverting)
The enzyme, found in animals and plants, is involved in the biosynthesis of O-fucosylated proteins. In EGF domains, the attachment of O-linked fucose to serine or threonine occurs within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys.
CAS REGISTRY NUMBER
COMMENTARY hide
9033-08-3
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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
GDP-beta-L-fucose + protein
GDP + ?
show the reaction diagram
GDP-beta-L-fucose + protein
GDP + ?
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
GDP-beta-L-fucose + protein
GDP + ?
show the reaction diagram
protein O-fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this protein O-fucosylation and selectively glycosylate specific residues in epidermal growth factor-like (EGF) repeats and thrombospondin type I repeats (TSRs). PoFUT1 glycosylates epidermal growth factor-like (EGF) repeats within the consensus sequence C2-X-X-X-X-S/T-C3
-
-
?
GDP-beta-L-fucose + protein
GDP + ?
show the reaction diagram
protein O-fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this protein O-fucosylation and selectively glycosylate specific residues in epidermal growth factor-like (EGF) repeats and thrombospondin type I repeats (TSRs). PoFUT2 glycosylates thrombospondin type I repeats (TSRs) containing Ser/Thr residues located in the consensus sequences C1-X-X-S/T-C2 or C2-X-X-S/T-C3 of TSRs of groups 1 and 2
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-
?
additional information
?
-
POFUT1s bind GDP-fucose and EGF repeats, and transfer this monosaccharide into small EGF repeats producing GDP during the reaction
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
activates transfer of fucose from DP-fucose to small EGF repeats
Mn2+
activates wild-type and mutant enzymes, but has a nonessential role in catalysis. Residue R290 replaces Mn2+ function by establishing electro­static and hydrogen bond interactions with beta-phosphate
additional information
Mg2+ is not required for catalytic activity by POFUT1, glycosyltransferases adopting GT-B folds are metal-independent
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
CePOFUT1 is a member of the GT65 family and contains four conserved disulfide bridges through the GT65 family
physiological function
protein O-fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this protein O-fucosylation and selectively glycosylate specific residues in epidermal growth factor-like (EGF) repeats and thrombospondin type I repeats (TSRs). PoFUT1 glycosylates epidermal growth factor-like (EGF) repeats within the consensus sequence C2-X-X-X-X-S/T-C3
physiological function
additional information
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
OFUT1_CAEEL
389
0
44051
Swiss-Prot
Secretory Pathway (Reliability: 3)
SUBUNIT
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of the enzyme in the unliganded form and in complex with GDP and GDP-fucose
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
crystal structures of the ternary complex between the enzyme and the human TSR1 repeat
purified recombinant enzyme CePOFUT2 in complex with GDP and human TSR1, X-ray diffraction structure determination and analysis
PROTEIN VARIANTS
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
G19Q
site-directed mutagenesis, the mutation affects both secretion and fucosylation
G19R
site-directed mutagenesis, the mutation affects both secretion and fucosylation
G20H
site-directed mutagenesis, the mutation affects both secretion and fucosylation
R298K/R299K
site-directed mutagenesis, the mutant enzyme is stable against proteolysis and similarly active as the wild-type. The mutant enzymes is capable of fucosylating TSRs not only of group 1 but also of group 2, it not only recognizes and reacts with TSRs showing slightly different structures but also accepts TSRs with very low sequence identity. Residue Glu52 of mutant CePOFUT is the catalytic base
S15D
site-directed mutagenesis, the mutant shows increased secretion, but a significant reduc­tion in fucosylation, suggesting that HsPOFUT2 is highly selective for amino acids in the Xa position
S15Q
site-directed mutagenesis, the mutant shows increased secretion, but a significant reduc­tion in fucosylation, suggesting that HsPOFUT2 is highly selective for amino acids in the Xa position
V16H
site-directed mutagenesis, the mutation affects both secretion and fucosylation
additional information
mutants R40A, N43A and R240A/K are more stable while F199A, D309N, D242A, D244A, W245A, F261A and F357A are less stable than the wild-type. Mutants R40A, R240A/K, W245A and F357A show a decrease in binding to GDP, from this group, R40A and W245A bind better to GDP than F357A, R240K and R240A, with the latter being impaired in binding
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
recombinant enzyme CePOFUT2 in complex with GDP and human TSR1 by gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
gene profut2, recombinant expression in Pichia pastoris and secretion to the medium, partially degradation of the protein
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lira-Navarrete, E.; Valero-Gonzalez, J.; Villanueva, R.; Martinez-Julvez, M.; Tejero, T.; Merino, P.; Panjikar, S.; Hurtado-Guerrero, R.
Structural insights into the mechanism of protein O-fucosylation
PLoS ONE
6
e25365
2011
Caenorhabditis elegans (Q18014)
Manually annotated by BRENDA team
Valero-Gonzalez, J.; Leonhard-Melief, C.; Lira-Navarrete, E.; Jimenez-Oses, G.; Hernandez-Ruiz, C.; Pallares, M.C.; Yruela, I.; Vasudevan, D.; Lostao, A.; Corzana, F.; Takeuchi, H.; Haltiwanger, R.S.; Hurtado-Guerrero, R.
A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2
Nat. Chem. Biol.
12
240-246
2016
Caenorhabditis elegans (Q8WR51), Caenorhabditis elegans
Manually annotated by BRENDA team
Lira-Navarrete, E.; Hurtado-Guerrero, R.
A perspective on structural and mechanistic aspects of protein O-fucosylation
Acta Crystallogr. Sect. F
74
443-450
2018
Caenorhabditis elegans (Q18014), Caenorhabditis elegans (Q8WR51), Mus musculus (Q91ZW2), Homo sapiens (Q9H488), Homo sapiens (Q9Y2G5)
Manually annotated by BRENDA team