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Literature summary for 1.14.13.113 extracted from

  • 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 (2013), Biochemistry, 52, 477-487.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli Klebsiella pneumoniae

Crystallization (Commentary)

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Klebsiella pneumoniae A6T923
-
-
Klebsiella pneumoniae ATCC 700721 A6T923
-
-

Purification (Commentary)

Purification (Comment) Organism
nickel affinity chromatography Klebsiella pneumoniae

Substrates and Products (Substrate)

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

Subunits Comment Organism
monomer
-
Klebsiella pneumoniae

Synonyms

Synonyms Comment Organism
flavin adenine dinucleotide-dependent urate oxidase
-
Klebsiella pneumoniae
HpxO
-
Klebsiella pneumoniae

Turnover Number [1/s]

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

Cofactor Comment Organism Structure
FAD
-
Klebsiella pneumoniae
FAD dependent on Klebsiella pneumoniae

kcat/KM [mM/s]

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