Cloned (Comment) | Organism |
---|---|
gene glce, recombinant expression of enzyme mutant comprising residues Arg50-Asn585 as N-terminally His6-SUMO-tagged enzyme in Escherichia coli strain BL21(DE3), expression of N-terminally His6-SUMO-tagged SeMet-substituted Glce protein in Escherichia coli strain B834, expression of diverse enzyme point mutants | Danio rerio |
Crystallization (Comment) | Organism |
---|---|
purified enzyme in apo-form and in complex with heparin hexasaccharide, both in a stable dimer conformation, hanging drop vapour diffusion method, mixing of 0.001 ml of 15-20 mg/ml protein in in 20 mM Tris, pH 8.0, 200 mM ammonium acetate, 1 mM dithiothreitol, and 1 mM EDTA, with 0.001 ml of 16% w/v PEG 3350, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, and 2% v/v Tacsimate, pH 5.0 for the unlabeled enzyme and 16% w/v PEG 3350, 0.06 M citric acid, and 0.04 M Bis-Tris propane, pH 4.1, for the SeMet-labeled enzyme, a concentration of 5.0 mg/ml is used for the enzyme complex with heparin oligosaccharide at a molar ratio of 1:5, 20°C, 3 days, X-ray diffraction structure determination and analysis at 1.9-2.2 A resolution | Danio rerio |
Protein Variants | Comment | Organism |
---|---|---|
H584A | site-directed mutagenesis, the mutant shows 80% reduced activity compared to wild-type | Danio rerio |
N585A | site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type | Danio rerio |
R154A | site-directed mutagenesis, the mutant shows 60% reduced activity compared to wild-type | Danio rerio |
R156A | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
R396A | site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type | Danio rerio |
R531A | site-directed mutagenesis, the mutant shows about 90% reduced activity compared to wild-type | Danio rerio |
R543A | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y149F | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y177F | site-directed mutagenesis, the mutant shows 40% reduced activity compared to wild-type | Danio rerio |
Y179F | site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type | Danio rerio |
Y468A | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y468F | site-directed mutagenesis, the mutant shows 75% reduced activity compared to wild-type | Danio rerio |
Y482F | site-directed mutagenesis, the mutant shows 20% reduced activity compared to wild-type | Danio rerio |
Y528A | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y528F | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y546A | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Y546F | site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type | Danio rerio |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
heparin hexasaccharide | mechanism of product inhibition of the enzyme: 2-O- and 6-O-sulfation of heparan sulfate keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues, binding structure, detailed overview | Danio rerio |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
125000 | - |
recombinant enzyme, gel filtration | Danio rerio |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Danio rerio | F1QR43 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-SUMO-tagged enzyme mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, tag cleavage, another step of nickel affinity chromatography, and gel filtration of the eluate | Danio rerio |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | heparin hexasaccharide is product of Glce following O-sulfation, structure of the Glce dimer in complex with heparin hexasaccharide, detailed overview | Danio rerio | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 60600, recombinant enzyme, SDS-PAGE | Danio rerio |
More | the enzyme forms a dimer with two catalytic sites, each at a positively charged cleft in C-terminal alpha-helical domains binding one negatively charged hexasaccharide | Danio rerio |
Synonyms | Comment | Organism |
---|---|---|
D-glucuronyl C5-epimerase | - |
Danio rerio |
Glce | - |
Danio rerio |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Danio rerio |
General Information | Comment | Organism |
---|---|---|
metabolism | D-glucuronyl C5-epimerase is a crucial modifying enzyme in the heparan sulfate biosynthesis pathway | Danio rerio |
additional information | enzyme structure-function relationship, active site structure and function, overview. Three tyrosine residues, Tyr468, Tyr528, and Tyr546, in the active site are crucial for the enzymatic activity | Danio rerio |
physiological function | heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-Glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition | Danio rerio |