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Enzyme Nomenclature
Information on EC 1.14.13.113 - FAD-dependent urate hydroxylase
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
1.14.13.113
-
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RECOMMENDED NAME
GeneOntology No.
FAD-dependent urate hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
urate + NADH + H+ + O2 = 5-hydroxyisourate + NAD+ + H2O
a flavoprotein. The product 5-hydroxyisourate is spontaneously converted to allantoin
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
urate,NADH:oxygen oxidoreductase (5-hydroxyisourate-forming)
A flavoprotein. The reaction is part of the purine catabolic pathway in the bacterium Klebsiella pneumoniae. The enzyme is different from EC 1.7.3.3, factor-independent urate hydroxylase, found in most plants, which produces hydrogen peroxide. The product of the enzyme is a substrate for EC 3.5.2.17, hydroxyisourate hydrolase.
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
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-
?
additional information
?
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urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
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-
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urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
part of the purine catabolic pathway
-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
part of the purine catabolic pathway
-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
part of the purine catabolic pathway
unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
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?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
urate hydroxylase shows selectivity (V/K ratio of 10) for NADH over NADPH
unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
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?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
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-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
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-
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
Q8PDQ6
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
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-
?
additional information
?
-
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urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
-
-
-
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
B5B0J6
part of the purine catabolic pathway
-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
B5B0J6
part of the purine catabolic pathway
-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
part of the purine catabolic pathway
unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
A6T923
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-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
A6T923
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-
-
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Q8PDQ6
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
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-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADH
-
the enzyme shows selectivity (V/K ratio of 10) for NADH over NADPH
NADH
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
NADPH
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the enzyme shows selectivity (V/K ratio of 10) for NADH over NADPH
NADPH
the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.1
NADH
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant Y216F, pH 8.0, 25°C
0.5
NADH
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; wild-type, pH 8.0, 25°C
0.64
NADH
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant R204Q, pH 8.0, 25°C
0.042
Urate
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; wild-type, pH 8.0, 25°C
0.09
Urate
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.26
NADH
Klebsiella pneumoniae
A6T923
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C
8.4
NADH
Klebsiella pneumoniae
A6T923
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C
42
NADH
Klebsiella pneumoniae
A6T923
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C
0.26
Urate
Klebsiella pneumoniae
A6T923
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant R204Q, pH 8.0, 25°C
8.4
Urate
Klebsiella pneumoniae
A6T923
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant Y216F, pH 8.0, 25°C
42
Urate
Klebsiella pneumoniae
A6T923
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; wild-type, pH 8.0, 25°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1
NADH
Klebsiella pneumoniae
A6T923
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C
8
84
NADH
Klebsiella pneumoniae
A6T923
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant Y216F, pH 8.0, 25°C
8
125
NADH
Klebsiella pneumoniae
A6T923
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; wild-type, pH 8.0, 25°C
8
93
Urate
Klebsiella pneumoniae
A6T923
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; mutant Y216F, pH 8.0, 25°C
710
1000
Urate
Klebsiella pneumoniae
A6T923
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25°C; wild-type, pH 8.0, 25°C
710
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578)
Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578)
Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45000
-
recombinant enzyme including the affinity tag, determined by SDS-PAGE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
2 * 52000, SDS-PAGE; 2 * 52519, calculated from amino acid sequence
monomer
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 0.05 M KH2PO4 and 20% (w/v) PEG 8000; 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
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and heterologous overexpression as N-terminally six-His tagged protein
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R204Q
the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation; 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
Y216F
about 10% of wild-type catalytic efficiency; the mutant shows decreased activity compared to the wild type enzyme
R204Q
-
the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation; 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
-
Y216F
R204Q
-
the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation; 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
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Y216F
-
about 10% of wild-type catalytic efficiency; the mutant shows decreased activity compared to the wild type enzyme
-
Y216F
-
the mutant shows decreased activity compared to the wild type enzyme
-
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