Literature summary for 2.4.1.221 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.

Search for more literature for 2.4.1.221

Cloned(Commentary)

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 (Commentary)

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

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

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

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

Metals/Ions

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Caenorhabditis elegans	Q18014	-	-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

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

Synonyms	Comment	Organism
Pofut1	-	Caenorhabditis elegans
protein O-fucosyltransferase 1	-	Caenorhabditis elegans

Temperature Optimum [°C]

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

Temperature Stability [°C]

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

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

General Information

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