Information on EC 1.7.3.3 - factor-independent urate hydroxylase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea

EC NUMBER
COMMENTARY hide
1.7.3.3
-
RECOMMENDED NAME
GeneOntology No.
factor-independent urate hydroxylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
urate + O2 + H2O = 5-hydroxyisourate + H2O2
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
-
-
-
-
additional information
-
the enzyme catalyzes the degradation of urate to [S]-allantoin through 5-hydroxyisourate as a metastable intermediate
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
allantoin degradation
-
-
Caffeine metabolism
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
Purine metabolism
-
-
urate degradation to allantoin I
-
-
SYSTEMATIC NAME
IUBMB Comments
urate:oxygen oxidoreductase
This enzyme was previously thought to be a copper protein, but it is now known that the enzymes from soy bean (Glycine max), the mould Aspergillus flavus and Bacillus subtilis contains no copper nor any other transition-metal ion. The 5-hydroxyisourate formed decomposes spontaneously to form allantoin and CO2, although there is an enzyme-catalysed pathway in which EC 3.5.2.17, hydroxyisourate hydrolase, catalyses the first step. The enzyme is different from EC 1.14.13.113 (FAD-dependent urate hydroxylase).
CAS REGISTRY NUMBER
COMMENTARY hide
9002-12-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
NH-Rockefeller strain
UniProt
Manually annotated by BRENDA team
Aedes aegypti NH-Rockefeller
NH-Rockefeller strain
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
FERM BP-360
-
-
Manually annotated by BRENDA team
soil isolate
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
TB-90
-
-
Manually annotated by BRENDA team
TB-90
-
-
Manually annotated by BRENDA team
subspecies subtilis LMD 69.3
-
-
Manually annotated by BRENDA team
Candida sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
atlantic cod
Uniprot
Manually annotated by BRENDA team
partial sequence
DQ887577
GenBank
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Atlantic halibut
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain ZZJ4-1
-
-
Manually annotated by BRENDA team
strain ZZJ4-1
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
African lungfish
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
yeast-like fungi
endosymbiont of Nilaparvata lugens
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
maize
-
-
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
5-hydroxyisourate + O2
(S)-allantoin + H2O2 + CO2
show the reaction diagram
-
-
-
?
urate + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
uric acid + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
-
purine degradation
-
-
?
additional information
?
-
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 + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
uric acid + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
-
purine degradation
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
strong activation at 10 mM
CaCl2
-
enhances activity
Co2+
-
-
copper
Cu2+
-
enzyme contains copper, inhibited by excessive addition of Cu2+
Fe3+
-
activates
K+
-
slight activation at 10 mM
Mg2+
-
strong activation at 10 mM
Mn2+
2 mM, 170% of initial activity
Na+
-
slight activation at 10 mM
NaCl
-
enhances activity
NH4Cl
-
slight activation at 10 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,3,7-Trimethylxanthine
-
i.e. caffeine, slight
2,2'-dipyridyl
2,9-Dimethyl-1,10-phenanthroline
-
neo-cuproin
2-Hydroxypurine
-
-
3,7-Dimethylxanthine
-
i.e. theobromine, slight
3-Methyluric acid
-
-
5,5'-dithiobis(2-nitrobenzoate)
-
-
5-Azaorotate
7-Methyluric acid
-
-
8-Azaxanthine
8-nitroxanthine
-
-
9-methyluric acid
adenine
Allantoic acid
-
weak
allantoin
Amelide
-
-
-
arginine
-
weak
aspartic acid
-
slight
Ba2+
Candida sp.
-
-
beta-mercaptoethanol
-
about 60% residual activity after 1 h incubation with 0.5 mM beta-mercaptoethanol at pH 8.5 and 25C
Biguanidine salts
-
inactivation is pH-dependent: slightly inhibitory below pH 10, rapid inactivation at high pH
Cyanurate
-
-
D-sorbitol
-
about 70% residual activity after 1 h incubation with 0.5 mM D-sorbitol at pH 8.5 and 25C
Dicyandiamide
-
inactivation is pH-dependent: small below pH 10, rapid increase at high pH
diethyldithiocarbamic acid
-
-
Fe3+
Candida sp.
-
-
glutamine
glycerol
-
-
glyoxylic acid
-
weak
Guanidinium salts
-
inactivation is pH-dependent: slightly inhibitory below pH 10, rapid inactivation at high pH
hydroxylamine
-
-
Hydroxypurines
-
-
-
hypoxanthine
inosine 5'-monophosphate
-
-
iodoacetate
-
low effect
Li+
-
18.5% inhibition at 1 mM
N-ethylmaleimide
neocuproin
o-Iodosobenzoate
-
-
o-phenanthroline
oxonate
Oxopurines
-
-
-
p-chloromercuribenzoate
Periodate
-
-
phosphate
-
no inactivation by phosphate, in presence of borate or dithiothreitol
pyrazinoate
Salicylhydroxamic acid
-
-
SDS
-
49% inhibition at 0.5% w/v
Sodium deoxycholate
-
about 90% residual activity after 1 h incubation with 0.5 mM sodium deoxycholate at pH 8.5 and 25C
Thiourea
-
slight
Trichloropurine
xanthine
ZnCl2
-
1 mM, 91% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Uric acid
DQ887577
uric acid (0.3%) is an inducer for uricase production, concentrations higher than 0.3% do not enhance the enzyme productivity
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.029 - 0.061
O2
0.0000135 - 1.05
Urate
0.00588 - 1.5
Uric acid
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.01 - 18.1
Urate
31.3
Uric acid
Glycine max
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
510 - 2620
Urate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0021
8-nitroxanthine
-
competitive inhibitor versus urate at pH 8.0
0.008
oxonate
-
unmodified enzyme, 50 mM borate buffer, 25C, pH 9.2
0.041 - 4.5
xanthine
additional information
additional information
-
1-methylurate, 3-methylurate, 7-methylurate do not inhibit significantly
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.067
-
wild-type, pH 8.9, 25C
2.49
-
urate oxidase including p-azido-L-phenylalanine instead of Phe at position 281, in 0.1 M borate, pH 8.4
2.67
-
recombinant strain overexpressing the enzyme, pH 8.9, 25C
2.99
mutant A89T/G91A7V92M/H245L/E252A/M253I/R291K/A296V/A301S/K303R, pH 8.6, 25C
3.74
-
cotyledons, 4 d old
4.22
wild-type, pH 8.6, 25C
4.91
mutant H245L/E252A/M253I/R291K/A296V/A301S/K303R, pH 8.6, 25C
5.29
-
-
5.32
-
purified enzyme
5.68
mutant A296V, pH 8.6, 25C
5.94
mutant R291K/A296V/A301S/K303R, pH 8.6, 25C
6.85
-
-
8.26
-
urate oxidase including p-azido-L-phenylalanine instead of Phe at position 170, in 0.1 M borate, pH 8.4
9.35
-
male rats
10.5
-
in the cell lysate
12.2
Candida sp.
-
-
13.3
-
hypocotyls, 4 d old
15.3
-
female
15.4
-
roots, 4 d old
16.33
-
natural uricase, in 0.1 M borate, pH 8.4
18
-
crude extract, pH 8.0, 37C
21.5
-
after DEAE Sepharose FF chromatography
25.7
-
after Phenyl-Sepharose FF chromatography
27
-
after HiLoad 26/60 Superdex 75 gel filtration
38.4
recombinant protein, pH 8.0, 37C
39
-
after 2.1fold purification, pH 8.0, 37C
1790
-
nodules, 21 d old
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
-
yield of recombinant uricase is significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase
7
DQ887577
optimal pH for uricase production in culture flasks
8 - 8.5
-
borate and phosphate buffer
8.4
-
assay at
8.5 - 9.3
-
-
8.6
-
free enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9.5
-
free uricase shows at least 50% relative activity between pH 6.5 and 9.5, around pH 7.5, free uricase remains 81.16% of its maximum activity, while the uricase loaded in the lipid vesicles remains almost the same high activity (178.26%) as its optimum activity (179.72%)
6.5 - 11
-
pH 6.5: about 40% of maximal activity, pH 10.0: about 50% of maximal activity
7 - 11
-
pH 7: about 50% of activity maximum, pH 11: about 40% of activity maximum
7.4 - 9.6
-
50% of activity maximum at pH 7.4 and pH 9.6, free enzyme
7.5 - 8.5
-
-
8 - 10.2
-
pH 8.0: about 35% of activity maximum, pH 10.2: about 55% of activity maximum
8 - 11
-
50% of activity maximum at pH 8 and pH 11, immobilized enzyme
8.5 - 10.5
-
50% of activity maximum at pH 8.5 and pH 10.5
8.5 - 9.5
Candida sp.
-
-
8.6 - 9.5
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27
-
isoform UIV
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 50
-
20C: about 70% of activity maximum; 50C: about 60% of activity maximum
20 - 70
-
free uricase shows at least 50% relative activity between 20 and 70C
20 - 50
-
20C: about 60% of activity maximum; 50C: about 50% of activity maximum
20 - 55
-
20C: about 80% of maximal activity, maximal activity at 30C, 55C: about 55% of maximal activity
35 - 60
-
-
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.96
-
isoelectric focusing, pH gradient 3.5-9.5
7.6
-
isoelectric focusing, rasburicase
8.46
-
determined by means of P/ACE 5000
9.38
calculation from nucleotide sequence
additional information
-
isoelectric focusing presents a trail of pI values between 6.55 and 7.6
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
strain CGMCC 2.120 is used as source of uricase gene
Manually annotated by BRENDA team
-
wild-type and PEX5-defective CHO cell lines each stable producing the enzyme
Manually annotated by BRENDA team
-
no uricase is detected in mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. Uricase activity is increased 10fold to 40fold under derepression conditions and is induced by exogenous uric acid
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
the enzyme is mainly localized in the membrane of PEX5-defective mutant cells
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus sp. (strain TB-90)
Bacillus sp. (strain TB-90)
Bacillus sp. (strain TB-90)
Bacillus sp. (strain TB-90)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000 - 35000
-
SDS-PAGE, gel filtration
32880
yeast-like fungi
-
deduced from nucleotide sequence of cDNA
33270
-
determination of nucleotide sequence of cDNA and calculation of corresponding amino acid sequence
35050
-
deduced from nucleotide sequence of cDNA
35060
-
MALDI-TOF mass spectrometry
35780
-
MALDI-TOF mass spectrometry
35790
-
calculated from amino acid sequence
50000
-
gel filtration
58680
-
MALDI-TOF mass spectrometry
58900
-
calculated from amino acid sequence
60000
-
about 60000 Da, SDS-PAGE
68000
-
gel filtration
70000 - 76000
Candida sp.
-
gel filtration
100000
102000
-
polyacrylamide disc electrophoresis
105000
-
gel filtration
109000
-
gel filtration
114000 - 128000
-
PAGE, gel filtration
115000 - 123000
-
PAGE, gel filtration
117000
-
gel filtration
120000 - 122000
120000 - 140000
-
PAGE, gel filtration
120000
-
gel filtration, equilibrium sedimentation
124000
125000
128000
136300 - 141600
-
ultracentrifugation
137000
-
for the homotetramer, determined by neutron crystallographic analysis
140000
Candida sp.
-
gel filtration
144000
-
native enzyme, gel filtration
145000 - 150000
-
gel filtration
151000
-
Sephadex G-200 gel filtration
230000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
hexamer
-
alpha6, 6 * 37000, SDS-PAGE
homotetramer
monomer
tetramer
trimer
-
alpha3, 3 * 32000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallizations are performed using the hanging-drop vapour-diffusion method at 19.9C, structures of crystals soaked with the substrate uric acid, the inhibitor 8-azaxanthin and allantoin are determined at 1.9-2.2 A resolution, 2 homotetramers comprise the asymmetric crystallographic unit, each subunit contains 2 T-fold domains of topology, which are usually found in purine- and pterin-binding enzymes, the uric acid substrate is bound tightly to the enzyme by interactions with Arg180, Leu222 and Gln223 from one subunit and with Thr67 and sp68 of the neighbouring subunit in the tetramer
; Rasburicase
-
crystallization of large proteins in the presence of polyethylene glycol
-
crystals of about a few tens of micrometres in size, which is nucleated previously in crystallization batch containing 5% PEG 8000, 100 mM NaCl, 8 mg/ml uox-substrate complex and 100 mM Tris-HCl pH 8.5, are used as seeds and their size and quality are further improved using a temperature-control device, large crystals of Uox, co-crystallized with its substrates analogues 8-azaxanthine, 9-methyluric acid or the natural substrate in the presence of cyanide (0.5-2 mg/ml), and soaks with the natural substrate in the absence of cyanide, diffracting to high resolutions are obtained, in the presence of different inhibitors, the crystal form of Uox has a body-centred orthorhombic symmetry and one of the largest primitive unit-cell volumes (a: 80 A, b: 96 A, c: 106 A)
-
enzyme in complex with substrate urate and inhibitor cyanide, X-ray diffraction structure determination and analysis
-
hanging drop vapour diffusion method
-
ligand-free Uox crystallized with NH4Cl and 15% (w/v) PEG 8000, ligand-free Uox crystallized in water with 10% (w/v) PEG 8000, ligand-free Uox crystallized with NaCl and 15% (w/v) PEG 8000, ligand-free Uox crystallized with (NH4)2SO4 and 15% (w/v) PEG 8000, ligand-free Uox crystallized with NaCl and 8% PEG 8000, ligand-free Uox crystallized with KCl and 10% (w/v) PEG 8000, and Uox complexed with 8-azaxanthine and crystallized with NaCl and 10% (w/v) PEG 8000, in 50 mM Tris buffer pH 8.0
-
recombinant enzyme in complex with inhibitor 8-azaxanthine in presence of O2 or Cl-, batch technique at room temperature, 10-15 mg/ml protein with an excess of 0.5-2 mg/ml of 8-azaxanthin in 50 mM Tris/HCl, pH 8.5, in the presence of 5-8% w/v PEG 8000 and 0.05 M NaCl, 24-48 h, X-ray diffraction structure determination and analysis at 1.6-1.7 A resolution
-
sitting drop vapour diffusion method
-
sitting drop vapour diffusion method using buffered D2O
-
sitting-drop vapour-diffusion method at room temperature
-
sitting-drop vapour-difusion method. Four different crystal forms of Uox are analyzed. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit of which contains one tetramer of Uox. Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per asymmetric unit. Coexistence of two crystal forms, P2(1) with two tetramers per asymmetric unit and I222, is found in the same crystallization drop containing another inhibitor, guanine. A fourth form, P2(1)2(1)2 with one tetramer per asymmetric unit, results in the presence of cymelarsan, an additive
-
homology modeling of monomeric enzyme. The highly conserved residue Gly290 could interact with Asn262 and His264. Residue substitutions near Gly290 may affect its spatial orientation and result in changes in catalysis.Gly290 is likely to participate in the structure of the active site and to be involved in oxygen-binding
to 1.93 A resolution. Space group P212121 with unit cell parameters a 69.16 A, b 139.31 A, c 256.33 A, and alpha =beta =gamma =90
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
-
35C, 1 h, stable
394144
6.5 - 10.5
-
22C, 30 min, immobilized enzyme, stable
394156
7
-
UOX is deactivated at different protein concentrations at 45C in 20 mM phosphate containing 0.15 M NaCl, 0.01 mg/ml UOX is deactivated much faster than its counterparts at concentrations of 0.1 and 1.0 mg/ml
700631
7 - 11
-
10 min, stable
394178
7 - 9.5
-
in case of uricase entrapped in lipid vesicles, the remaining activity keeps more than 90% during the pH of 7.0-9.5, and the maximum remaining activity is 98.04% at pH 8.0 when incubated at 40C or 40 min. For the free uricase, the maximum remaining activity is 86.59% at pH 8.5 when incubated at 40C or 40 min
712197
7 - 10
-
purified enzyme, 25C, 18 h, stable
701416
8 - 11
-
4C, 60 days, 35% loss of activity
394132
8 - 9
-
45C, 30 min, stable
394146
8.5 - 11
-
stable
659535
10
-
45C, 30 min, 30% loss of activity
394146
11
-
25C, 18 h, about 60% loss of activity
701416
12
-
22C, 30 min, 55% loss of activity
394156
additional information
-
uricase activity in 50 h culture broth with pH values of 5.5 and 6.0 decreases more rapidly than that in cultures with pH values of 6.5 and 7.0, at pH 5.5, about 78% of initial uricase activity is lost within 25 h, under the same conditions, more than 85% of initial uricase activity remains in culture broth of pH 6.5 and 7.0, uricase activity in 66 h culture broth with pH 7.0 degrades much more rapidly than that in samples from 50 h culture, while for pH 6.5, the uricase is still stable, loss of uricase activity is caused by the degradation in acidic environment by proteases secreted by the host cells or releases from host cell lyses, low pH may cause instability of uricase
695795
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 80
-
about 80% relative activity at 10C, about 85% relative activity at 20-30C, about 90% relative activity at 37C, about 40% relative activity at 40C, about 30% relative activity at 50C, about 20% relative activity at 60C, about 10% relative activity at 70C, and no activity at 80C after 30 min incubation
18 - 20
-
2 days, 70% loss of activity
45
Candida sp.
-
after 5 min 50% of initial activity
52
-
uricase covalently linked to monomethoxypoly(ethylene glycol) N-leucine-OSu-uricase and branched monomethoxypoly(ethylene glycol) N-leucine-OSu-uricase 50% loss of activity
55
-
uricase entrapped in lipid vesicles at enzyme concentrations of 0.005 and 0.1 mg/ml shows no loss of activity after 5 h at 55C. Uricase entrapped in lipid vesicles at enzyme concentrations of 0.01 mg/ml shows about 35% loss of activity after 5 h at 55C. Uricase entrapped in lipid vesicles at enzyme concentrations of 0.005 mg/ml shows about 50% loss of activity after 5 h at 55C. The free uricase at 0.005 mg/ml is rapidly deactivated to about 30% of the initial activity within an incubation time of 2 h, while more than 70% of the initial enzyme activity remains for the uricase at 0.1 mg/ml in the identical incubation time
60
-
10 min, stable
60 - 61
-
native uricase and poly(N-acryloylmorpholine)-OSu-uricase 50% loss of activity
65
-
30 min, the purified enzyme retains 98.9% of its original activity
70
-
30 min, the purified enzyme retains 64% of its original activity
75
DQ887577
enzyme is thermostable, after heat treatment at 75C for 45 min, the uricase retains about 100% of its initial activity
80
DQ887577
70% of initial activity remains after 45 min at 80C
100
-
35% relative activity at 100C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
after modification with monomethoxy-poly(ethylene glycol)-5000, the recombinant intracellular uricase shows residual activity of about 65%
-
ammonium sulfate protects from inactivation at low pH
-
at the physiological pH, significant increase of enzyme activity is found for the uricase entrapped in the lipid vesicles (1.8times that of free uricase) at their respective optimum pH. Free uricase shows rapid decrease in its enzymatic activity with a half life of less than 20 min when incubated with trypsin. Uricase entrapped in the lipid vesicles gradually loses its activity but still 50% of the original activity remains after 60 min (remaining activity is 7.32% in case of free uricase)
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borate stabilizes
conjugated uricases are more stable to trypsin digestion
-
Cu2+ inactivates at low temperature, uric acid prevents inactivation
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dithiothreitol effect of treatment with dithiothreitol on extraction and purification
-
dithiothreitol prevents polymerization and stabilizes throughout purification
-
dithiothreitol stabilization
-
EDTA stabilizes
Fe3+ partially protects against inactivation at low pH or at low ionic strength, stimulates reactivation
-
frozen enzyme retains complete activity
-
little loss of activity by freeze-drying
-
lower stability in solutions of phosphate buffer than in borate buffer
-
proteolytic digestion by endopeptidases cause rapid loss of activity, exopeptidases have slight effect
Repeated freezing and thawing has no effect
-
stability of immobilized enzyme depends on the time of stirring during immobilization and on the quantity of enzyme used
-
unusually resistant to guanidinium chloride
-
unusually resistant to SDS
-
urate oxidase from female rat livers is more stable than enzyme from male rat livers
-
urea, 4 M, several h without loss of activity
-
uricase is reversibly inactivated in solutions of low ionic strength (like during dialysis against water). After incubation for 2 h in 100 mM sodium chloride in water at 4C, the dialysis-inactivated uricase shows about 70% of the maximal specific activity. After incubation for 2 h in 100 mM Tris-HCl pH 8.0 plus 100 mM NaCl at 4C, the dialysis-inactivated uricase shows about 90% of the maximal specific activity. After pre-incubation for 0.5 h in sodim borate plus 100 mM NACL at 25C followed by the direct addition of urate to measure its activity, the dialysis-inactivated uricase shows about 80% of the maximal specific activity
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DMSO
-
the residual recombinant UOX activity in the presence of DMSO is significantly higher than that in pure H2O, the residual UOX activity increases in response to the increase in the DMSO concentration up to 20%, further increase in DMSO concentration (50-70%) results in significant UOX deactivation
Methanol
-
the residual recombinant UOX activity in the presence of methanol is significantly higher than that in pure H2O, the residual UOX activity increases in response to the increase in the methanol concentration up to 20%, further increase in methanol concentration (50-70%) results in significant UOX deactivation
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
sensitive to O2
-
246662
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-12C, 0.10 mol/l Tris/HCl buffer (pH 8.0), 8 months, less than 20% loss of activity, enzyme remains stable
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-15C, 100 mM borate buffer, pH 8.0, containing 100 mM or more ammonium sulfate
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-20C, 72 h, no loss of activity
-20C, purified enzyme, 72 h, 20% loss of activity
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0-4C, crystals in (NH4)2SO4 solution, 3 months, stable
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37C, purified enzyme, 12 h and 48 h, 50% and 100% loss of activity
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3C, 0.15% sodium carbonate, several weeks, stable
-
4C, 0.10 mol/l Tris/HCl buffer (pH 8.0), 10 weeks, less than 20% loss of activity
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4C, 100 mM Tris-HCl buffer, pH 9.5, 90% of activity after 5 days
yeast-like fungi
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4C, 20 mM phosphate buffer, pH 7.8, for at least 1 month
-
4C, 7 days, 40% loss of activity
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4C, 72 h, no loss of activity
4C, free uricase in borate buffer (pH 8.5), 28 days, 70% loss of activity
-
4C, purified enzyme, 72 h, 20% loss of activity
-
4C, several weeks
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4C, uricase in lipid vesicles in borate buffer (pH 8.5), 28 days, no loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, ion exchange chromatography and gel filtration
-
by anion exchange and hydropbobic interaction chromatography
by Ni-NTA affinity chromatography
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DEAE Sepharose FF chromatography, Phenyl-Sepharose FF chromatography and HiLoad 26/60 Superdex 75 gel filtration
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DEAE-cellulose column chromatography
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enzyme synthesized with C-terminal 6-histidine tag
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native enzyme 19.7fold by ammonium sulfate fractionation, anion exchange exchange and hydrophobic interaction chromatography, and gel filtration
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Ni-NTA column chromatography
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recombinant enzyme
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Superdex S200PG gel filtration
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uricase-MBP fusion protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a plasmid containing the AgUOX gene is introduced into the expression host cell Escherichia coli DH1
complete cDNA and genomic DNA fragment coding for urate oxidase is isolated and characterized, heterologous expression in Escherichia coli
developing of a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system, substitution of p-azido-L-phenylalanine for F170 or F281 in urate oxidase, optimization of the system by adding a Shine-Dalgarno sequence and tandem suppressor tRNA in order to increase the expression levels of tyrosyl suppressor tRNA and aminoacyl-tRNA synthetase
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expressed in Chinese hamster ovary cells
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expressed in Escherichia coli
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expressed in Escherichia coli BL21 (DE3)
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expressed in Escherichia coli BL21 (DE3) cells
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expressed in Escherichia coli BL21(DE3) cells
expressed in Saccharomyces cerevisiae
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expression in Escherichia coli
expression in Escherichia coli DH5alpha
yeast-like fungi
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expression in Escherichia coli MC1061
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expression in Escherichia coli RR1
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expression in Escherichia coli, Escherichia coli harboring pUOD1 produces 20fold higher uricase than the original Arthrobacter strain, even without an inducer
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expression in NM 538
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expression in pPUO1
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expression in Saccharomyces cerevisiae
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overexpression in Escherichia coli JM109
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overexpression in Pichia angusta
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uricase production by the recombinant Hansenula polymorpha strain MU200 harboring Candida utilis uricase gene under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence
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using the recombinant DNA technique, enzyme is obtained from a genetically modified Saccharomyces cerevisiae strain that expresses urate oxidase cDNA, cloned from a strain of Aspergillus flavus
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D79A
-
the KM-value for urate is 82% of the wild-type value
F179A
-
decreases Vmax by 2 orders of magnitude
F179Y
-
mutation decreases Vmax by 2-fold
T69A
-
mutant enzyme has a maximal velocity of 3% of the wild-type value. Ionization at pH 6.4 that is observed with the wild-type enzyme is absent in the mutant. The KM-value for urate is 5fold higher than that of the wild-type enzyme
T69A/K9M
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the KM-value for urate is 16.1fold higher than that of the wild-type enzyme
T69V
-
the KM-value for urate is 30.9fold higher than that of the wild-type enzyme
A296V
about 50% ioncrease in catalytic efficiency
A89T/G91A7V92M/H245L/E252A/M253I/R291K/A296V/A301S/K303R
replacement with the corresponding residues of human enzyme. About 30% of wild-type catalytic efficiency
H245L/E252A/M253I/R291K/A296V/A301S/K303R
replacement with the corresponding residues of human enzyme. Catalytic efficiency is higher than in wild-type, but below the efficiency of mutant R291K/A296V/A301S/K303R
I115V/H119R/L120F/H245L/E252A/M253I/R291K/A296V/A301S/K303R
replacement with the corresponding residues of human enzyme, complete loss of activity
R119H
complete loss of activity
R291K/A296V/A301S/K303R
replacement with the corresponding residues of human enzyme. About 50% increase in catalytic efficiency
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
drug development
-
urate oxidase has the potential to be a therapeutic target for the treatment of gout
medicine
pharmacology
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urate oxidase is a potential therapeutic protein in the prevention and treatment of tumor lysis syndrome and hyperuricemia. However, its severe immunogenicity limits its clinical application. Engineering site-specific modifications of keto groups in urate oxidase by using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s)/suppressor tRNA pairs reduces its antigenicity. The mutated uricase exhibits decreased antigenic properties, while its catalytic activities remain unchanged
synthesis
high-yield expression of uricase in Escherichia coli and establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein is more than 98% and the specific activity is 38.4 IU/mg