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Information on EC 1.14.13.113 - FAD-dependent urate hydroxylase and Organism(s) Klebsiella pneumoniae and UniProt Accession A6T923

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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.
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This record set is specific for:
Klebsiella pneumoniae
UNIPROT: A6T923
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The taxonomic range for the selected organisms is: Klebsiella pneumoniae
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
urate hydroxylase, hpxo enzyme, fad-dependent urate oxidase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
flavin adenine dinucleotide-dependent urate oxidase
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FAD-dependent urate oxidase
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
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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.
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 + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
show the reaction diagram
additional information
?
-
-
urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
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-
?
NATURAL SUBSTRATE
NATURAL 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 + NADH + H+ + O2
5-hydroxyisourate + NAD+ + H2O
show the reaction diagram
-
part of the purine catabolic pathway
unstable in aqueous buffer, hydrolyzing spontaneously to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
-
?
additional information
?
-
-
urate oxidase can catalyze the slow conversion of NADPH to NADP+ in the absence of urate
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
-
the enzyme is a flavoprotein
NADH
-
the enzyme shows selectivity (V/K ratio of 10) for NADH over NADPH
NADPH
-
the enzyme shows selectivity (V/K ratio of 10) for NADH over NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 0.64
NADH
0.042 - 0.9
Urate
0.042
Urate
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pH 8.0, cosubstrate 1.5 mM NADH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.26 - 42
NADH
0.26 - 42
Urate
42
Urate
-
pH 8.0, 1.5 mM NADH as cosubstrate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 125
NADH
93 - 1000
Urate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
-
recombinant enzyme including the affinity tag, determined by SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R204Q
Y216F
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
nickel affinity chromatography
enzyme is purified by Ni2+-nitrilotriacetic acid chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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
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
Manually annotated by BRENDA team
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)
Manually annotated by BRENDA team