Information on EC 1.14.13.113 - FAD-dependent urate hydroxylase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.113
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RECOMMENDED NAME
GeneOntology No.
FAD-dependent urate hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
urate + NADH + H+ + O2 = 5-hydroxyisourate + NAD+ + H2O
show the reaction diagram
a flavoprotein. The product 5-hydroxyisourate is spontaneously converted to allantoin
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
urate degradation to allantoin II
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Purine metabolism
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Metabolic pathways
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Microbial metabolism in diverse environments
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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.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain M5al
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
show the reaction diagram
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
show the reaction diagram
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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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
show the reaction diagram
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
show the reaction diagram
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
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.024 - 0.64
NADH
0.055
NADPH
in 50 mM K2HPO4/NaH2PO4, pH 8.0, at 25C
0.029 - 0.9
Urate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19 - 42
NADH
1.14
NADPH
Xanthomonas campestris
Q8PDQ6
in 50 mM K2HPO4/NaH2PO4, pH 8.0, at 25C
0.26 - 42
Urate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 125
NADH
8
21
NADPH
Xanthomonas campestris
Q8PDQ6
in 50 mM K2HPO4/NaH2PO4, pH 8.0, at 25C
5
37 - 1000
Urate
710
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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recombinant enzyme including the affinity tag, determined by SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
2 * 52000, SDS-PAGE; 2 * 52519, calculated from amino acid sequence
monomer
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
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme is purified by Ni2+-nitrilotriacetic acid chromatography
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nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and heterologous overexpression as N-terminally six-His tagged protein
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expressed in Acinetobacter baylyi strain ADP1
expressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
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
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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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R204Q
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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
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about 10% of wild-type catalytic efficiency; the mutant shows decreased activity compared to the wild type enzyme
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