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Literature summary extracted from

  • 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 (2011), PLoS ONE, 6, e25365.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.221 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 Caenorhabditis elegans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.221 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 Caenorhabditis elegans

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 R240A site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant Caenorhabditis elegans
2.4.1.221 R240K site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans
2.4.1.221 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 Caenorhabditis elegans

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.1.221 endoplasmic reticulum
-
 Caenorhabditis elegans 5783
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.4.1.221 Mn2+ activates transfer of fucose from DP-fucose to small EGF repeats Caenorhabditis elegans
2.4.1.221 additional information Mg2+ is not required for catalytic activity by POFUT1, glycosyltransferases adopting GT-B folds are metal-independent Caenorhabditis elegans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.1.221 additional information Caenorhabditis elegans POFUT1s bind GDP-fucose and EGF repeats, and transfer this monosaccharide into small EGF repeats producing GDP during the reaction ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.221 Caenorhabditis elegans Q18014
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.221 recombinant truncated POFUT1_26-382 from Pichia pastoris by affinity and ion exchange chromatography and gel filtration Caenorhabditis elegans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.221 additional information POFUT1s bind GDP-fucose and EGF repeats, and transfer this monosaccharide into small EGF repeats producing GDP during the reaction Caenorhabditis elegans ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.1.221 More 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 Caenorhabditis elegans

Synonyms

EC Number Synonyms Comment Organism
2.4.1.221 Pofut1
-
 Caenorhabditis elegans
2.4.1.221 protein O-fucosyltransferase 1
-
 Caenorhabditis elegans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.221 25
-
 assay at Caenorhabditis elegans

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.4.1.221 50 55 dependent on ligands present, Mn2+ shifts the optimum to 50°C, GDP to 55°C, overview Caenorhabditis elegans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.221 7.5
-
 assay at Caenorhabditis elegans

General Information

EC Number General Information Comment Organism
2.4.1.221 evolution CePOFUT1 is a member of the GT65 family and contains four conserved disulfide bridges through the GT65 family Caenorhabditis elegans
2.4.1.221 additional information GDP-fucose is bound in a conserved cavity formed mainly by amino acids from the C-terminal domain, it is localised in the interface where the two domains face each other, localisation of EGF repeat binding site in CePOFUT1, active site and ligand binding structure analysis, detailed overview Caenorhabditis elegans