Information on EC 1.6.3.3 - NADH oxidase (H2O2-forming)

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

EC NUMBER
COMMENTARY hide
1.6.3.3
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RECOMMENDED NAME
GeneOntology No.
NADH oxidase (H2O2-forming)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NADH + H+ + O2 = NAD+ + H2O2
show the reaction diagram
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
non-pathway related
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SYSTEMATIC NAME
IUBMB Comments
NADH:oxygen oxidoreductase (H2O2-forming)
A flavoprotein (FAD). The bacterium Streptococcus mutans contains two distinct NADH oxidases, a H2O2-forming enzyme and a H2O-forming enzyme (cf. EC 1.6.3.4, NADH oxidase (H2O-forming)) [1]. The enzymes from the anaerobic archaea Methanocaldococcus jannaschii [6] and Pyrococcus furiosus [3] also produce low amounts of H2O. Unlike EC 1.6.3.1 (NAD(P)H oxidase) it has no activity towards NADPH.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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the lack of a significant effect on deletion of the genes from Streptococcus mutans suggests the presence of additional antioxidant proteins in this bacterium
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 NADH + H+ + O2
2 NAD+ + 2 H2O
show the reaction diagram
the enzyme produces both H2O and H2O2. 62% of NADH-derived reducing equivalents are recovered as H2O2 and the rest probably generates H2O. The NADPH oxidase activity is about 5% compared to the activity with NADH
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-
?
2 NADH + H+ + O2
NAD+ + 2 H2O
show the reaction diagram
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the enzyme produces both H2O and H2O2, It is highly specific for NADH, little or no activity with NADPH. NOX1 produces 23% water and 77% H2O2 as products under the assay conditions given
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-
?
beta-NADH + H+ + O2
beta-NAD+ + H2O2
show the reaction diagram
NADH + H+ + O2
NAD+ + H2O2
show the reaction diagram
NADPH + H+ + O2
NAD+ + H2O2
show the reaction diagram
the enzyme produces both H2O and H2O2. 62% of NADH-derived reducing equivalents are recovered as H2O2 and the rest probably generates H2O. The NADPH oxidase activity is about 5% compared to the activity with NADH
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-
?
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
beta-NADH + H+ + O2
beta-NAD+ + H2O2
show the reaction diagram
NADH + H+ + O2
NAD+ + H2O2
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CuCl2
10 mM, 3-4fold stimulation
K3Fe(CN)6
10 mM, 3-4fold stimulation
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-chloromercuribenzoate
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0.1 mM, 40% inhibition
5,5'-dithiobis-(2-nitrobenzoate)
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1 mM, 7 min, 50% loss of activity
AgNO3
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1 mM, 63% inhibition
ascorbate
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1 mM, 81% inhibition
Ba2+
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0.1 mM, 86% inhibition
Ca2+
-
0.1 mM, about 20% inhibition
CaCl2
10 mM, slightly increases activity
Cd(CH3CH2COO-)2
10 mM, slightly increases activity
Co2+
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0.1 mM, 31% inhibition
CoCl2
10 mM, slightly increases activity
Cr2+
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0.1 mM, 49% inhibition
Cu2+
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0.1 mM, complete inhibition
CuCl2
CuSO4
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1 mM, 1 h at 22°C, 85% remaining activity
cysteine
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1 mM, 54% inhibition
dithiothreitol
2 mM, rapid decrease in activity to less than 10% of the activity
EGTA
10 mM, slightly increases activity
Fe2+
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0.1 mM, 56% inhibition
FeSO4
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1 mM, 1 h at 22°C, 56% remaining activity
Guanidine-HCl
Hg2+
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0.1 mM, complete inhibition
HgCl2
hydrocortisone
-
3 mM, 5% inhibition
MgCl2
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1 mM, 1 h at 22°C, 93% remaining activity
Mn2+
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0.1 mM, about 20% inhibition
MnCl2
10 mM, slightly increases activity
Ni2+
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0.1 mM, 73% inhibition
NiCl2
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1 mM, 1 h at 22°C, 71% remaining activity
Quinacrine
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3 mM, 46% inhibition
Quinine
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3 mM, 23% inhibition
SDS
0.5%, completely inhibits the reaction
Sn2+
-
0.1 mM, complete inhibition
Sodium deoxycholate
0.5%, slightly decreases activity
Zn2+
-
0.1 mM, 53% inhibition
ZnCl2
ZnSO4
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1 mM, 1 h at 22°C, 69% remaining activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
250 mM, 10fold activation
2-mercaptoethanol
2 mM, stimulates up to 2fold
CHAPS
0.2-0.5%, increases activity about twofold
dithiothreitol
FAD
flavoprotein, addition of 0.06 mM results in 3.7fold stimulation; flavoprotein, Addition of 0.06 mM results in about 2.5fold stimulation
n-dodecyl beta-D-maltoside
0.5%, increases activity about twofold
p-chloromercuribenzoate
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1 mM, 1.9fold activation
Triton X-100
0.1-0.5%, increases activity about twofold
Tween 20
1.25%, increases activity about twofold
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0037
beta-NADH
pH and temperature not specified in the publication
0.00153 - 0.13
NADH
0.043 - 2.9
O2
additional information
additional information
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Km for NADH is below 4 mM, whereas the substrate-level FAD-dependent portion of the activity shows a Km for FAD of 0.044 mM. kcat for the oxidase reaction in the absence of substrate-level FAD is 4.8/s, while kcat for the reaction in the presence of substrate-level FAD is 11.1/s
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
115
beta-NADH
Pyrococcus horikoshii
O58049
pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2
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pH 8.8, 25°C
30
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pH 7.0, 80°C
37
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30°C, pH not specified in the publication
84
pH and temperature not specified in the publication
144
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pH 7.0, 80°C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4 - 8.4
pH 4.4: about 70% of maximal activity, pH 8.4: about 90% of maximal activity
4.5 - 9.8
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the activity decreases with pH in the range pH 4.5 to 9.8
4.5 - 8
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pH 4.5: about 90% of maximal activity, pH 8.0: about 50% of maximal activity
5 - 8
pH 5.0: about 50% of maximal activity, pH 8.0: about 50% of maximal activity
5.5 - 8.5
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pH 5.5: 73% of maximal activity (50 mM Mes buffer), pH 8.5: 81% of maximal activity (50 mM Mops buffer)
6 - 9.5
pH 6.0: about 60% of maximal activity, pH 9.5: about 60% of maximal activity
6.5 - 9
pH 6.5: about 55% of maximal activity, pH 9.0: about 55% of maximal activity
6.5 - 7.5
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very active in the pH-range 6.5-7.5
7.9 - 11
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pH 7.8: about 60% of maximal activity, pH 11.0: about 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
30 - 58
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the activity increases with the temperature, as tested from 30°C to 58°C
75
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22 - 90
active from room temperature to 90°C. At 50°C, the activity of the recombinant enzyme is half that at 80°C
30 - 70
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activity at 30°C is about 40% of the maximal at 70°C
30 - 50
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30°C; about 75% of maximal activity, 50°C: about 80% of maximal activity
55 - 100
55°C: about 60% of maximal activity, 100°C: about 80% of maximal activity
60 - 90
60°C: about 50% of maximal activity, 90°C: about 95% of maximal activity; 60°C: about 50% of maximal activity, 90°C: about 95% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6
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isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22000
4 * 22000, SDS-PAGE
27000
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1 * 27000
35000
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2 * 35000
46000
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1 * 54000 + 1 * 46000, SDS-PAGE
47000
x * 47000, SDS-PAGE
48000
2 * 48000, the enzyme exists as a dimer and to some extent as a tetramer, SDS-PAGE; 4 * 48000, the enzyme exists as a dimer and to some extent as a tetramer, SDS-PAGE
54000
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1 * 54000 + 1 * 46000, SDS-PAGE
55196
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x * 55196, calculated from sequence
56000
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4 * 56000, SDS-PAGE
57000
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2 * 57000, SDS-PAGE
68000
1 * 68000, the enzyme exists as a monomer and to some extent as a dimer, SDS-PAGE; 2 * 68000, the enzyme exists as a monomer and to some extent as a dimer, SDS-PAGE
84000
gel filtration
90000
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gel filtration
94000
dimeric enzyme form, gel filtration
97100
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gel filtration
100000
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gel filtration
102000
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gel filtration
152000
tetrameric enzyme form, gel filtration
178000
tetrameric enzyme form, gel filtration
220000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
monomer
tetramer
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
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37°C, 1 h, the enzyme retains full activity at pH 7.0, but activity declines following incubation at either acidic or alkaline pH
722881
6 - 10
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1 h, stable
721365
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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pH 7.0, 1 h, enzyme retains full activity
55
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pH 7.0, 1 h, activity markedly decreases
60
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30 min, enzyme retains 5% of its activity
83
pH 7.6, 40 min, the half-life of the recombinant enzyme is 12 min, the partially purified native enzyme has a half-life of 35 h
95
half-life: 4 h
98
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half-life: 77 min
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
apparently, the enzyme in the cell extract is more resistant to oxygen inactivation than the purified enzyme. Purified enzyme samples exposed to air exhibit a decrease in enzyme activity. The inactivation rate of NADH oxidase activity is dependent on oxygen concentration. The times required for the loss of 50% of the enzyme activity from the purified enzyme are about 20 min and 40 min for oxygen concentrations of 20% (v/v) and 1% (v/v), respectively. However, the times required for the loss of 50% of the enzyme activity from the cell extract aree about 60 min and 360 min for oxygen concentration of 20% (v/v) and 1% (v/v), respectively
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722518
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 6 months, enzyme retains full activity
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4°C. pH 7.0, 1 week, activity decreases by 80%
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichi coli C41
expression in Escherichia coli
expression in Escherichia coli; expression in Escherichia coli
expression of the nox-1 gene in Escherichia coli using its own promoter
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overexpressed in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
aerobically induced
expressed constitutively under strictly anaerobic conditions. The fact that the expression of the Nox enzymes is not regulated suggests that they have some fundamental metabolic role, and not an occasional role during oxygen stress; expressed constitutively under strictly anaerobic conditions. The fact that the expression of the Nox enzymes is not regulated suggests that they have some fundamental metabolic role, and not an occasional role during oxygen stress
transcriptional analysis demonstrates that NOX1 is constitutively expressed regardless of the carbon source and a single promoter is identified 25 bp upstream of the nox1 gene by primer extension
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis