Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli | Klebsiella pneumoniae |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapor diffusion method, using 0.05 M KH2PO4 and 20% (w/v) PEG 8000 | Klebsiella pneumoniae |
structures of enzyme with and without uric acid at 2.0 and 2.2 A, respectively, and of the R204Q variant at 2.0 A resolution in the absence of uric acid. The variant structure is very similar to that of wild-type HpxO except for the conformation of Arg103, which interacts with FAD in the variant but not in the wild-type structure. The R204Q variant results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid, activating it for the oxygen transfer | Klebsiella pneumoniae |
Protein Variants | Comment | Organism |
---|---|---|
R204Q | variant results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid, activating it for the oxygen transfer | Klebsiella pneumoniae |
R204Q | the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation | Klebsiella pneumoniae |
Y216F | the mutant shows decreased activity compared to the wild type enzyme | Klebsiella pneumoniae |
Y216F | about 10% of wild-type catalytic efficiency | Klebsiella pneumoniae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.042 | - |
Urate | wild-type, pH 8.0, 25°C | Klebsiella pneumoniae | |
0.042 | - |
Urate | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.09 | - |
Urate | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.1 | - |
NADH | mutant Y216F, pH 8.0, 25°C | Klebsiella pneumoniae | |
0.1 | - |
NADH | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.5 | - |
NADH | wild-type, pH 8.0, 25°C | Klebsiella pneumoniae | |
0.5 | - |
NADH | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.64 | - |
NADH | mutant R204Q, pH 8.0, 25°C | Klebsiella pneumoniae | |
0.64 | - |
NADH | mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.9 | - |
Urate | mutant Y216F, pH 8.0, 25°C | Klebsiella pneumoniae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
urate + NADH + H+ + O2 | Klebsiella pneumoniae | - |
5-hydroxyisourate + NAD+ + H2O | - |
? | |
urate + NADH + H+ + O2 | Klebsiella pneumoniae ATCC 700721 | - |
5-hydroxyisourate + NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Klebsiella pneumoniae | A6T923 | - |
- |
Klebsiella pneumoniae ATCC 700721 | A6T923 | - |
- |
Purification (Comment) | Organism |
---|---|
nickel affinity chromatography | Klebsiella pneumoniae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
urate + NADH + H+ + O2 | - |
Klebsiella pneumoniae | 5-hydroxyisourate + NAD+ + H2O | - |
? | |
urate + NADH + H+ + O2 | - |
Klebsiella pneumoniae ATCC 700721 | 5-hydroxyisourate + NAD+ + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | - |
Klebsiella pneumoniae |
Synonyms | Comment | Organism |
---|---|---|
flavin adenine dinucleotide-dependent urate oxidase | - |
Klebsiella pneumoniae |
HpxO | - |
Klebsiella pneumoniae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.26 | - |
Urate | mutant R204Q, pH 8.0, 25°C | Klebsiella pneumoniae | |
0.26 | - |
NADH | mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
0.26 | - |
Urate | mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
8.4 | - |
Urate | mutant Y216F, pH 8.0, 25°C | Klebsiella pneumoniae | |
8.4 | - |
NADH | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
8.4 | - |
Urate | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
42 | - |
Urate | wild-type, pH 8.0, 25°C | Klebsiella pneumoniae | |
42 | - |
NADH | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
42 | - |
Urate | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Klebsiella pneumoniae | |
FAD | dependent on | Klebsiella pneumoniae |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.1 | - |
NADH | mutant R204Q, pH 8.0, 25°C | Klebsiella pneumoniae | |
1 | - |
NADH | mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
84 | - |
NADH | mutant Y216F, pH 8.0, 25°C | Klebsiella pneumoniae | |
84 | - |
NADH | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
93 | - |
Urate | mutant Y216F, pH 8.0, 25°C | Klebsiella pneumoniae | |
93 | - |
Urate | mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
125 | - |
NADH | wild-type, pH 8.0, 25°C | Klebsiella pneumoniae | |
125 | - |
NADH | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae | |
1000 | - |
Urate | wild-type, pH 8.0, 25°C | Klebsiella pneumoniae | |
1000 | - |
Urate | wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C | Klebsiella pneumoniae |