Cloned (Comment) | Organism |
---|---|
dicDHPR gene (residues 1-231) cloned into pET-28b expression plasmid with hexahistidine tag and thrombin cleavage site, expression in Escherichia coli BL21 (DE3) | Dictyostelium discoideum |
Crystallization (Comment) | Organism |
---|---|
dicDHPR-NAD complex by incubation in 20 mM Tris-HCl, pH 8.0, hanging-drop vapor diffusion (4 microl drops), 18°C (291 K), screening solution (0.1 M bis-tris, pH 6.5, 17% PEG 3350), hexahistidine tag not removed, x-ray diffraction | Dictyostelium discoideum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
24600 | - |
calculated, monomer | Dictyostelium discoideum |
25000 | - |
SDS-PAGE, monomer | Dictyostelium discoideum |
50000 | - |
gel-filtration chromatography (Superdex 200 column), dimer | Dictyostelium discoideum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
quinonoid dihydrobiopterin + NADH + H+ | Dictyostelium discoideum | - |
6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin + NAD+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Dictyostelium discoideum | - |
- |
- |
Purification (Comment) | Organism |
---|---|
cells centrifuged, washed with PBS (pH 7.4), resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5), disrupted by sonication, centrifugation, supernatant filtered (0.45 microm), applied to a column with nickel-NTA beads, eluted (50 mM Tris-HCl, pH 7.5), active fractions pooled, concentrated, and exchanged into 50 mM sodium phosphate, pH 6.8, ultrafiltration, applied to a cation-exchange chromatography HS20 column, eluted with salt gradient at pH 6.8, concentration by ultrafiltration, gel-filtration chromatography with Superdex 200 column in 50 mM Tris-HCl, pH 8.0, concentration of eluted fractions in 20 mM Tris-HCl, pH 8.0, by ultrafiltration | Dictyostelium discoideum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
quinonoid dihydrobiopterin + NADH + H+ | - |
Dictyostelium discoideum | 6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin + NAD+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 25000, four molecules form 2 dimers in the asymmetric unit in crystals | Dictyostelium discoideum |
Synonyms | Comment | Organism |
---|---|---|
dicDHPR | - |
Dictyostelium discoideum |
dihydropteridine reductase | - |
Dictyostelium discoideum |
General Information | Comment | Organism |
---|---|---|
metabolism | enzyme catalyzes the last step of tetrahydrobiopterin recycling | Dictyostelium discoideum |