Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes | Rhodobacter capsulatus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type enzyme and subunit beta mutants E232A and E232Q with or without molybdenum cofactor, hanging drop vapor diffusion method, 15 mg/ml protein in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl,and 2.5 mM dithiothreitol are mixed 1 mM NAD+ and inhibitor pterin-6-aldehyde and with the reservoir solution containing 6-8 mM BaCl2, 6-8% polyethylene glycol 8000, 100 mM Tris-HCl, pH 8.3, 5-25mM dithiothreitol, and 3-4% isopropanol, whereas the EB232Q variant is mixed in a 1:2 ratio with the same reservoir solution, X-ray diffraction structure determination and analysis at 2.6-3.4 A resolution, overview | Rhodobacter capsulatus |
Protein Variants | Comment | Organism |
---|---|---|
EB232Q | catalytically inactive active site mutant, inactive desulfo enzyme form | Rhodobacter capsulatus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
allopurinol | i.e. 1-H-pyrazolo [3,4-d] pyrimidine-4-one | Rhodobacter capsulatus | |
oxypurinol | - |
Rhodobacter capsulatus | |
pterin-6-aldehyde | competitive inhibition pattern, mechanism of inhibitor binding at the active site, overview | Rhodobacter capsulatus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | - |
Rhodobacter capsulatus | 5829 | - |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
hypoxanthine + NAD+ + H2O | Rhodobacter capsulatus | - |
xanthine + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | Rhodobacter capsulatus | - |
urate + NADH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodobacter capsulatus | O54050 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes with or without molybdenum cofactor by nickel affinity and anion exchange chromatography, followed by gel filtration | Rhodobacter capsulatus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
xanthine + NAD+ + H2O = urate + NADH + H+ | the catalytic sequence of Rhodobacter capsulatus XDH is initiated by abstraction of a proton from the Mo-OH group by the highly conserved active site subunit B residue Glu730, followed by nucleophilic attack of the resulting Mo-O on the carbon center of the substrate (C-2 in hypoxanthine and C-8 in xanthine) and concomitant hydride transfer to the Mo-S of the molybdenum center, reaction and substrate binding mechanisms, overview | Rhodobacter capsulatus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
hypoxanthine + NAD+ + H2O | - |
Rhodobacter capsulatus | xanthine + NADH + H+ | - |
? | |
hypoxanthine + NAD+ + H2O | mechanism of substrate binding at the active site, importance of beta subunit residue Glu232 for substrate positioning, overview. The oxygen atom at the C-6 position of both substrates is oriented toward ArgB-310 in the active site | Rhodobacter capsulatus | xanthine + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | - |
Rhodobacter capsulatus | urate + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | mechanism of substrate binding at the active site, importance of beta subunit residue Glu232 for substrate positioning, overview. The oxygen atom at the C-6 position of both substrates is oriented toward ArgB-310 in the active site | Rhodobacter capsulatus | urate + NADH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
tetramer | (alphabeta)2 heterotetramer | Rhodobacter capsulatus |
Synonyms | Comment | Organism |
---|---|---|
XDH | - |
Rhodobacter capsulatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Rhodobacter capsulatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Rhodobacter capsulatus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Rhodobacter capsulatus |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.001 | - |
oxypurinol | - |
Rhodobacter capsulatus | |
0.1036 | - |
pterin-6-aldehyde | pH 7.5, 25°C | Rhodobacter capsulatus |