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Information on EC 1.14.13.113 - FAD-dependent urate hydroxylase
for references in articles please use BRENDA:EC1.14.13.113
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EC Tree 1 Oxidoreductases
1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
1.14.13 With NADH or NADPH as one donor, and incorporation of one atom of oxygen into the other donor
1.14.13.113 FAD-dependent urate hydroxylase
IUBMB Comments
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.
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
urate hydroxylase, hpxo enzyme, fad-dependent urate oxidase, more
top
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
flavin adenine dinucleotide-dependent urate oxidase
HpxO
HpxO enzyme
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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 SOURCE
PATHWAYS
MetaCyc
urate conversion to allantoin II
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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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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
Substrates: the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
Products: -
Products: -
?
additional information
?
-
-
Substrates: urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: part of the purine catabolic pathway
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: part of the purine catabolic pathway
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
Substrates: part of the purine catabolic pathway
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
Substrates: urate hydroxylase shows selectivity (V/K ratio of 10) for NADH over NADPH
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
urate + NADPH + H+ + O2
5-hydroxyisourate + NADP+ + H2O
Substrates: the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
Products: -
Products: -
?
additional information
?
-
-
Substrates: urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: part of the purine catabolic pathway
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: part of the purine catabolic pathway
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
-
Substrates: part of the purine catabolic pathway
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
Products: unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
urate + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
Substrates: the enzyme is slightly more efficient (about 2.6times) with NADPH than NADH
Products: -
Products: -
?
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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
-
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 25Ā°C
0.5
NADH
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
0.64
NADH
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
0.042
Urate
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
0.09
Urate
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.26
NADH
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
8.4
NADH
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
42
NADH
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
0.26
Urate
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
8.4
Urate
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
42
Urate
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1
NADH
mutant enzyme R204Q, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
84
NADH
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
125
NADH
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
93
Urate
mutant enzyme Y216F, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
1000
Urate
wild type enzyme, in 0.1 M potassium phosphate buffer (pH 8.0), at 25Ā°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Highest Expressing Human Cell Lines
Cell Line Links Show AA Sequence and Transmembrane Helices (471 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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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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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R204Q
Y216F
R204Q
Y216F
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
R204Q
the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation
Y216F
the mutant shows decreased activity compared to the wild type enzyme
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
-
R204Q
-
the mutation leading to strongly decreased activity results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation
-
Y216F
-
the mutant shows decreased activity compared to the wild type enzyme
-
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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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pope, S.D.; Chen, L.L.; Stewart, V.
Purine utilization by Klebsiella oxytoca M5al: genes for ring-oxidizing and -opening enzymes
J. Bacteriol.
191
1006-1017
2008
Klebsiella oxytoca (B5B0J6), Klebsiella oxytoca M5al (B5B0J6)
brenda
O'Leary, S.E.; Hicks, K.A.; Ealick, S.E.; Begley, T.P.
Biochemical characterization of the HpxO enzyme from Klebsiella pneumoniae, a novel FAD-dependent urate oxidase
Biochemistry
48
3033-3035
2009
Klebsiella pneumoniae
brenda
Hicks, K.A.; OLeary, S.E.; Begley, T.P.; Ealick, S.E.
Structural and mechanistic studies of HpxO, a novel flavin adenine dinucleotide-dependent urate oxidase from Klebsiella pneumoniae
Biochemistry
52
477-487
2013
Klebsiella pneumoniae (A6T923), Klebsiella pneumoniae, Klebsiella pneumoniae ATCC 700721 (A6T923)
brenda
Michiel, M.; Perchat, N.; Perret, A.; Tricot, S.; Papeil, A.; Besnard, M.; de Berardinis, V.; Salanoubat, M.; Fischer, C.
Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO
Environ. Microbiol. Rep.
4
642-647
2012
Xanthomonas campestris (Q8PDQ6), Xanthomonas campestris
brenda
EXTERNAL LINKS (specific for EC-Number 1.14.13.113)
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Release 2023.1 (January 2023)
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