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Information on EC 1.7.3.3 - factor-independent urate hydroxylase and Organism(s) Aspergillus flavus and UniProt Accession Q00511

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IUBMB Comments
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).
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Aspergillus flavus
UNIPROT: Q00511
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Word Map
The taxonomic range for the selected organisms is: Aspergillus flavus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Reaction Schemes
Synonyms
uricase, rasburicase, urate oxidase, uricase ii, uric acid oxidase, uricozyme, aguox, nodulin 35, elitek, fasturtec, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Rasburicase
Fasturtec
-
trade name of rasburicase
N-35
-
-
-
-
Nodule specific uricase
-
-
-
-
Nodulin 35
-
-
-
-
Nodulin 35 homolog
-
-
-
-
Non-symbiotic uricase
-
-
-
-
oxidase, urate
-
-
-
-
Rasburicase
Urate oxidase
uric acid oxidase
-
-
-
-
uricase
Uricoenzyme
-
-
Uricozyme
-
trade name of urate oxidase from Aspergillus flavus
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
urate + O2 + H2O = 5-hydroxyisourate + H2O2
show the reaction diagram
catalytic mechanism, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
-
oxidation
reduction
-
-
-
-
additional information
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
-
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
urate + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
additional information
?
-
anion-pi interactions are present in the active site of the enzyme and are energetically favorable
-
-
?
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 + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
urate + O2 + H2O
5-hydroxyisourate + H2O2
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
the catalytic mechanism of UOX does not imply any cofactor
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
the catalytic mechanism of UOX does not imply any metal ion
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8-Azaxanthine
CN-
the presence of residue Phe159 enhances the interaction energy of the anion with the urate pi system
8-Azaxanthine
9-methyluric acid
-
substrate analogue
hydroxylamine
-
-
Periodate
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
H2O2
25°C, pH not specified in the publication
0.0472
Urate
Rasburicase
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.3
H2O2
25°C, pH not specified in the publication
18.1
Urate
Rasburicase
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10.5
-
in the cell lysate
21.5
-
after DEAE Sepharose FF chromatography
25.7
-
after Phenyl-Sepharose FF chromatography
27
-
after HiLoad 26/60 Superdex 75 gel filtration
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 25
-
the enzyme is highly active in the range of 15-25°C. The enzyme loses approximately 35% of its original activity at temperatures over 35°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
calculated and isoelectric focusing
7.6
isoelectric focusing, rasburicase
8.46
-
determined by means of P/ACE 5000
additional information
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
URIC_ASPFL
302
0
34241
Swiss-Prot
other Location (Reliability: 3)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
62000
x * 62000, rasburicase, SDS-PAGE
13500
-
gel filtration
135000
137000
-
for the homotetramer, determined by neutron crystallographic analysis
33750
-
4 * 33750, X-ray crystallography
34000
-
SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 62000, rasburicase, SDS-PAGE
homotetramer
?
-
x * 35000, SDS-PAGE
homotetramer
tetramer
-
-
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with substrate urate and inhibitor cyanide, X-ray diffraction structure determination and analysis
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
quantum mechanical/molecular mechanical calculations based on PDB entry 4N9M. The oxidation consists of chemical transformation from 8-hydroxyxythine to an anionic radical via a proton transfer along with an electron transfer, proton transfer to the O2- anion (radical), diradical recombination to form a peroxo intermediate, and dissociation of H2O2 to generate the dehydrourate. Hydration is initiated by the nucleophilic attack of a water molecule on dehydrourate, along with a concerted proton transfer through residue Thr69 in the catalytic site. Hydration is the rate-determining step
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
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)
-
hanging 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
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
UOX is deactivated at different protein concentrations at 45°C 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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
12 h, 50% residual activity, complete loss of activity after 54 h
0 - 70
-
the enzyme remains stable (more than 90% activity) for 30 min at 0-20°C. The activity drops to about 60, 50, 40, less than 30, and less than 20% after 30 min at 30, 40, 50, 60, and 70°C, respectively. At 40°C, the enzyme shows about 80% activity after 5 min, about 65% activity after 10 min, about 50% activity after 20-40 min, and about 30% activity after 50-60 min
30 - 40
-
enzyme activity is diminished 60% when it is incubated at 30°C for 10 min. After 5 min at 40°C, there is a 25% decline in enzyme activity. The enzyme loses almost 60% of its original activity after 40 min at 40°C, whereas more than 50% of enzyme activity is preserved in the presence of lactose. Half-life at 40°C is almost 38 min and addition of raffinose does not change the half-life, whereas the presence of lactose has remarkable impact on enzyme half-life (46 min). Lactose notably enhances the melting temperature from 27 to 37°C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
lactose has protective effects on enzyme stability
-
proteolytic digestion by endopeptidases cause rapid loss of activity, exopeptidases have slight effect
-
the stability of the enzyme increases up to 4.9fold in the presence of 12% (w/v) of glycerol and sucrose. The effect of sorbitol on enzyme stability is negligible in the presence of glycerol and sucrose. In the presence of 20% (w/v) glycerol, sorbitol, and sucrose, the enzyme has the highest stability
-
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Superdex S200PG gel filtration
DEAE Sepharose FF chromatography, Phenyl-Sepharose FF chromatography and HiLoad 26/60 Superdex 75 gel filtration
-
Ni-NTA agarose affinity column chromatography
-
Ni-NTA agarose column chromatography, and Superdex 200 gel filtration
-
recombinant enzyme
-
Superdex S200PG gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3)
expressed in Saccharomyces cerevisiae
expression in Saccharomyces cerevisiae
expressed in Escherichia coli BL21 (DE3) cells
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Saccharomyces cerevisiae
-
expression in Saccharomyces cerevisiae
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
a colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters based on hydrogen peroxide quantitation. The general advantages of the colorimetric assay are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material
medicine
substitute for allopurinol in the management of gout and hyperuricaemia
synthesis
expression of the Escherichia-coli-codon-optimized gene as a fusion with the N-terminus of Mxe GyrA intein and chitin-binding domain for simple purification. After purification, the cleavage of the fusion protein is induced by adding DTT. Pure and properly folded uricase is obtained
medicine
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Conley, T.G.; Priest, D.G.
Non-classical inhibition of uricase by cyanide
Biochem. J.
187
733-738
1980
Aspergillus flavus
Manually annotated by BRENDA team
Fitzpatrick, D.A.; McGeeney, K.F.
Resistance of urate oxidase to proteolytic digestion
Biochem. Soc. Trans.
3
1253-1255
1975
Aspergillus flavus, Metabacillus fastidiosus, Cyberlindnera jadinii
-
Manually annotated by BRENDA team
Vivares, D.; Bonnete, F.
X-ray scattering studies of Aspergillus flavus urate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins
Acta Crystallogr. Sect. D
58
472-479
2002
Aspergillus flavus
Manually annotated by BRENDA team
Retailleau, P.; Colloc'h, N.; Vivares, D.; Bonnete, F.; Castro, B.; El-Hajji, M.; Mornon, J.P.; Monard, G.; Prange, T.
Complexed and ligand-free high-resolution structures of urate oxidase (Uox) from Aspergillus flavus: a reassignment of the active-site binding mode
Acta Crystallogr. Sect. D
60
453-462
2004
Aspergillus flavus
Manually annotated by BRENDA team
Retailleau, P.; Colloc'h, N.; Vivares, D.; Bonnete, F.; Castro, B.; El Hajji, M.; Prange, T.
Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site
Acta Crystallogr. Sect. D
61
218-229
2005
Aspergillus flavus
Manually annotated by BRENDA team
Fraisse, L.; Bonnet, M.C.; de Farcy, J.P.; Agut, C.; Dersigny, D.; Bayol, A.
A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters
Anal. Biochem.
309
173-179
2002
Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Bayol, A.; Capdevielle, J.; Malazzi, P.; Buzy, A.; Claude Bonnet, M.; Colloc'h, N.; Mornon, J.P.; Loyaux, D.; Ferrara, P.
Modification of a reactive cysteine explains differences between rasburicase and uricozyme, a natural Aspergillus flavus uricase
Biotechnol. Appl. Biochem.
36
21-31
2002
Aspergillus flavus, Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Budayova-Spano, M.; Bonnete, F.; Ferte, N.; El Hajji, M.; Meilleur, F.; Blakeley, M.P.; Castro, B.
A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin
Acta crystallogr. Sect. F
62
306-309
2006
Aspergillus flavus
Manually annotated by BRENDA team
Colloch, N.; Girard, E.; Dhaussy, A.; Kahn, R.; Ascone, I.; Mezouar, M.; Fourme, R.
High pressure macromolecular crystallography: The 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly
Biochim. Biophys. Acta
1764
391-397
2006
Aspergillus flavus
Manually annotated by BRENDA team
Gabison, L.; Chiadmi, M.; Colloch, N.; Castro, B.; El Hajji, M.; Prange, T.
Recapture of [S]-allantoin, the product of the two-step degradation of uric acid, by urate oxidase
FEBS Lett.
580
2087-2091
2006
Aspergillus flavus (Q00511), Aspergillus flavus
Manually annotated by BRENDA team
Li, J.; Chen, Z.; Hou, L.; Fan, H.; Weng, S.; Xu, C.; Ren, J.; Li, B.; Chen, W.
High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli
Protein Expr. Purif.
49
55-59
2006
Aspergillus flavus
Manually annotated by BRENDA team
Colloc'h, N.; Gabison, L.; Monard, G.; Altarsha, M.; Chiadmi, M.; Marassio, G.; Sopkova-de Oliveira Santos, J.; El Hajji, M.; Castro, B.; Abraini, J.H.; Prange, T.
Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism
Biophys. J.
95
2415-2422
2008
Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Gabison, L.; Prange, T.; Colloch, N.; El Hajji, M.; Castro, B.; Chiadmi, M.
Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: mechanistic implications
BMC Struct. Biol.
8
32
2008
Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Oksanen, E.; Blakeley, M.P.; Bonnete, F.; Dauvergne, M.T.; Dauvergne, F.; Budayova-Spano, M.
Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase
J. R. Soc. Interface
6 Suppl 5
599-610
2009
Aspergillus flavus
Manually annotated by BRENDA team
Liu, Z.; Lu, D.; Li, J.; Chen, W.; Liu, Z.
Strengthening intersubunit hydrogen bonds for enhanced stability of recombinant urate oxidase from Aspergillus flavus: molecular simulations and experimental validation
Phys. Chem. Chem. Phys.
11
333-340
2009
Aspergillus flavus (Q00511), Aspergillus flavus
Manually annotated by BRENDA team
Collings, I.; Watier, Y.; Giffard, M.; Dagogo, S.; Kahn, R.; Bonnete, F.; Wright, J.P.; Fitch, A.N.; Margiolaki, I.
Polymorphism of microcrystalline urate oxidase from Aspergillus flavus
Acta Crystallogr. Sect. D
66
539-548
2010
Aspergillus flavus (Q00511), Aspergillus flavus
Manually annotated by BRENDA team
Estarellas, C.; Frontera, A.; Quinonero, D.; Deya, P.
Relevant anion-π interactions in biological systems: The case of urate oxidase
Angew. Chem. Int. Ed. Engl.
50
415-418
2011
Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Wei, D.; Huang, X.; Qiao, Y.; Rao, J.; Wang, L.; Liao, F.; Zhan, C.
Catalytic mechanisms for cofactor-free oxidase-catalyzed reactions reaction pathways of uricase-catalyzed oxidation and hydration of uric acid
ACS Catal.
7
4623-4636
2017
Aspergillus flavus (Q00511)
Manually annotated by BRENDA team
Alishah, K.; Asad, S.; Khajeh, K.; Akbari, N.
Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme
Enzyme Microb. Technol.
93-94
92-98
2016
Aspergillus flavus (Q00511), Aspergillus flavus
Manually annotated by BRENDA team
Digumarti, R.; Sinha, S.; Nirni, S.S.; Patil, S.G.; Pedapenki, R.M.
Efficacy of rasburicase (recombinant urate oxidase) in the prevention and treatment of malignancy-associated hyperuricemia an Indian experience
Indian J. Cancer
51
180-183
2014
Aspergillus flavus
Manually annotated by BRENDA team
Imani, M.; Shahmohamadnejad, S.
Recombinant production of Aspergillus flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose
3 Biotech
7
201
2017
Aspergillus flavus
Manually annotated by BRENDA team
Mirzaeinia, S.; Pazhang, M.; Imani, M.; Chaparzadeh, N.; Amani-Ghadim, A.
Improving the stability of uricase from Aspergillus flavus by osmolytes Use of response surface methodology for optimization of the enzyme stability
Process Biochem.
94
86-98
2020
Aspergillus flavus
-
Manually annotated by BRENDA team