Cloned (Comment) | Organism |
---|---|
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 (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
R240A | site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant | Caenorhabditis elegans |
R240K | site-directed mutagenessis, altered mutant temperature dependence and GDP-fucose/GDP dissociation constants compared to the wild-type, inactive mutant | Caenorhabditis elegans |
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 |
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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
endoplasmic reticulum | - |
Caenorhabditis elegans | 5783 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | activates transfer of fucose from DP-fucose to small EGF repeats | Caenorhabditis elegans | |
additional information | Mg2+ is not required for catalytic activity by POFUT1, glycosyltransferases adopting GT-B folds are metal-independent | Caenorhabditis elegans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Caenorhabditis elegans | Q18014 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant truncated POFUT1_26-382 from Pichia pastoris by affinity and ion exchange chromatography and gel filtration | Caenorhabditis elegans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
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 | Comment | Organism |
---|---|---|
Pofut1 | - |
Caenorhabditis elegans |
protein O-fucosyltransferase 1 | - |
Caenorhabditis elegans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Caenorhabditis elegans |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | 55 | dependent on ligands present, Mn2+ shifts the optimum to 50°C, GDP to 55°C, overview | Caenorhabditis elegans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Caenorhabditis elegans |
General Information | Comment | Organism |
---|---|---|
evolution | CePOFUT1 is a member of the GT65 family and contains four conserved disulfide bridges through the GT65 family | Caenorhabditis elegans |
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 |