Information on EC 1.4.3.11 - L-glutamate oxidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.4.3.11
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RECOMMENDED NAME
GeneOntology No.
L-glutamate oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-glutamate + O2 + H2O = 2-oxoglutarate + NH3 + H2O2
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
-
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oxidative deamination
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-
-
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redox reaction
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-
-
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reduction
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SYSTEMATIC NAME
IUBMB Comments
L-glutamate:oxygen oxidoreductase (deaminating)
A flavoprotein (FAD).The enzyme from Azotobacter previously listed under this number, which did not produce H2O2, was a crude cell-free extract that probably contained catalase.
CAS REGISTRY NUMBER
COMMENTARY hide
39346-34-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the precursor of LGOX has a homodimeric structure and is less active than the mature enzyme with an alpha2beta2gamma2 structure, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-benzyl-L-glutamate + O2 + H2O
4-benzyl-2-oxoglutarate + NH3 + H2O2
show the reaction diagram
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13% of the activity with L-glutamate
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-
?
L-alanine + O2 + H2O
?
show the reaction diagram
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slight catalytic activity with the micro L-glutamate sensor with recombinant l-GlOx
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-
?
L-Asp + O2 + H2O
2-oxosuccinamic acid + NH3 + H2O2
show the reaction diagram
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at 0.6% of the activity with L-glutamate, at pH 7.4, no measurable activity at pH 6.0
-
-
?
L-aspartate + O2 + H2O
2-oxoglutarate + NH3 + H2O2
show the reaction diagram
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0.6% activity compared to L-glutamate
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-
?
L-aspartate + O2 + H2O
2-oxosuccinamic acid + NH3 + H2O2
show the reaction diagram
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slight catalytic activity with the micro L-glutamate sensor with recombinant l-GlOx
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-
?
L-Gln + O2 + H2O
2-oxoglutaric acid 5-amide + NH3 + H2O2
show the reaction diagram
L-glutamate + O2 + H2O
2-oxoglutarate + NH3 + H2O2
show the reaction diagram
L-glutamine + O2 + H2O
? + NH3 + H2O2
show the reaction diagram
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-
-
-
?
L-His + O2 + H2O
2-oxo-3-(1H-imidazol-4yl)-propionic acid + NH3 + H2O2
show the reaction diagram
L-Tyr + O2 + H2O
3-(4-hydroxyphenyl)-2-oxopropionic acid + NH3 + H2O2
show the reaction diagram
additional information
?
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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
L-glutamate + O2 + H2O
2-oxoglutarate + NH3 + H2O2
show the reaction diagram
additional information
?
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no activity with L-Leu, L-Phe, L-Asn, L-His, and L-Arg
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazol
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1 mM, 18% loss of activity
N-bromosuccinimide
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1 mM, 77.6% inhibition
p-aminobenzyloxanylic acid
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non-competitive
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.7 - 10
L-Gln
0.173 - 5
L-glutamate
5
L-His
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pH 6.8
additional information
L-glutamate
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KM for surface immobilised GluOx increases systematically with enzyme loading, due in part to electrostatic repulsion between the anionic substrate and neighbouring enzyme molecules at neutral pH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
27 - 85.8
L-glutamate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
308
L-glutamate
Streptomyces sp.
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wild type enzyme, pH and temperature not specified in the publication
41
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0105
p-aminobenzyloxanylic acid
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pH 5.6, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the enzyme exhibits 6.25 units/mg specific activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
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6
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immobilized L-GLOD
7
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recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
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pH 4.0: about 50% of maximal activity pH 10.0: about 40% of maximal activity
4 - 7
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pH 4.0: about 75% of maximal activity, pH 7.0: about 80% of maximal activity
5.5 - 8.5
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pH 5.5: about 30% of maximal activity, pH 8.5: about 80% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24
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immobilized L-GLOD, significant decrease in the optimum temperature when L-GLOD is bound to the membrane of the biosensor
35
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at pH 7.4, recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
50
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at pH 6.0, recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
58
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at pH 6.0 and at pH 7.4, recombinant enzyme treated with metalloendopeptidase from Streptomyces griseus
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 60
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25C: about 75% of maximal activity, 60C: about 55% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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recombinant enzyme from expression in Escherichia coli
Manually annotated by BRENDA team
additional information
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wheat bran culture
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70000
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mature LGOX SDS-PAGE and mass spectroscopy
90000
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gel filtration
95000
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gel filtration, non-denaturing PAGE
140000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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1 * 40000, 1 * 17000, and 1 * 12000, SDSPAGE, recombinant enzyme
hexamer
monomer
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1 * 75000, pre-mature enzyme, SDS-PAGE and MALDI-TOF mass spectrometry
tetramer
additional information
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the precursor of LGOX has a homodimeric structure and is less active than the mature enzyme with an alpha2beta2gamma2 structure, structure determination, overview. LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site, amino acid sequence/primary structure analysis, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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LGOX is expressed as a single polypeptide precursor in an incompletely active form. It forms a mature enzyme with a hexameric alpha2beta2gamma2 structure formed through protease modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme, sitting drop vapor diffusion method, mixing of protein solution containing 10 mg/mL protein in 20 mM KPB, pH 7.4, with 5 m; dithiothreitol, with a reservoir solution containing 1.2 M NaH2PO4, 0.8 M K2HPO4, 200 mM LiSO4, 100 mM CAPS, pH 6.2, in a 1:2 ratio, 5C, 3-4 weeks, X-ray diffraction structure determination and analysis at 2.6 A resolution
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crystals of mature LGOX are grown at 5C using the sitting drop vapor diffusion method. The LGOX crystals are formed in the presence of alpha-ketoglutarate. LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of a structurally homologous model of L-amino acid oxidase (LAAO) from snake venom reveales that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. The arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 7
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37C, 1 h, stable
391730
4.5 - 9.5
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60 min, 37C, stable
654238
5.5 - 7.5
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37C, 15 min, stable
391729
5.5 - 10.5
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45C, 15 min, stable
391731
6
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30C, 30 min, 50% inactivation, recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
655925
7.4
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50C, 30 min, 50% inactivation, recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
655925
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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pH 7.4, 30 min, 50% inactivation, recombinant enzyme before proteolysis by metalloendopeptidase from Streptomyces griseus
37
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pH 5.0-10.5, 1 h, stable
80
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pH 6.0, 30 min, 50% inactivation, recombinant enzyme treated with metalloendopeptidase from Streptomyces griseus
85
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pH 5.5, 15 min, 50% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C or -20C, pH 7.4, stable for several month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE Toyopearl 650 column chromatography
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recombinant enzyme from Escherichia coli strain JM109
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single-step purification with 95% recovery
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli strain JM109
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expression in Escherichia coli. The recombinant enzyme exhibits low thermostability compared to the enzyme isolated from Streptomyces sp. X-119-6 and is an aggregated form. Proteolysis of the recombinant enzyme with metalloendopeptidase from Streptomyces griseus improves the catalytic efficiency at various pH-values and stabilizes the structure of the enzyme
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mutant enzymes are expressed in Escherichia coli JM109 cells
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overexpression in Escherichia coli JM109
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H312A
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids
R305A
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids (best substrate is L-histidine)
R305D
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids (best substrate is L-arginine)
R305K
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids (best substrate is L-histidine)
R305L
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids (best substrate is L-histidine)
W564A
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the activity of the mutant enzyme toward L-glutamate is significantly reduced, however it exhibits catalytic activity toward various L-amino acids
additional information
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construction of a highly sensitive and selective glutamate microbiosensor based on polypyrrole, multiwalled carbon nanotubes, and glutamate oxidase deposited on the transducer platinum electrode, i.e. Pt/PPy/MWCNT/GluOx electrodes, overview. The biosensor has a high sensitivity, low response to interferences such as ascorbic acid, uric acid and acetaminophen, a fast response time, low detection limit, a linear range of 0.14 mM, and a satisfactory stability, mass transfer-limiting outer membrane of polyurethane, optimization, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
an amperometric microbiosensor for real time monitoring L-glutamate release in neural tissue, based on enzymatic oxidation catalyzed by the L-glutamate oxidase is developed. By means of a sol-gel coating method, L-glutamate oxidase is entrapped in a biocompatible gel layer that provides a benign environment and retains enzyme activity on the surface of Pt microelectrode. Prior to gel layer formation, a modification on the surface of Pt microelectrode with poly(phenylene diamine) enables the microbiosensor screen majority of common potential interfering substances existing in physiological samples. The resulting L-glutamate microbiosensors are characterized by a fast response, high sensitivity, favourable selectivity and excellent long-term stability
food industry
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development of a polarographic biosensor for monosodium glutamate by immobilizing L-GLOD on polycarbonate membrane and attached with oxygen electrode with a push cap system, usage of the membrane for over 20 measurements, showing stability
medicine