Information on EC 1.6.3.4 - NADH oxidase (H2O-forming)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.6.3.4
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RECOMMENDED NAME
GeneOntology No.
NADH oxidase (H2O-forming)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 NADH + 2 H+ + O2 = 2 NAD+ + 2 H2O
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
NADH:oxygen oxidoreductase (H2O-forming)
A flavoprotein (FAD). The bacterium Streptococcus mutans contains two distinct NADH oxidases, a H2O-forming enzyme and a H2O2-forming enzyme (cf. EC 1.6.3.3, NADH oxidase (H2O2-forming)) [1].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SwissProt
Manually annotated by BRENDA team
no activity in Clostridium acetobutylicum
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
the inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid biosynthesis. NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs fatty acid biosynthesis and hence blocks growth in aerobiosis
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 + 2 H+ + O2
2 NAD+ + 2 H2O
show the reaction diagram
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-
-
?
2 NADH + H+ + O2
NAD+ + 2 H2O
show the reaction diagram
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
2 NADH + H+ + O2
NAD+ + 2 H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
0.1 mM, 130% stimulation
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
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1 mM, 45% inhibition
Ca2+
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0.1 mM, about 20% inhibition
Cr2+
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0.1 mM, 92% inhibition
Cu2+
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0.1 mM, complete inhibition
FAD
optimum FAD concentration for the LrNox is 0.01 mM, with 90% and 3% of maximum activity at 0.001 mM and 0.5 mM FAD, respectively. Enzyme activity decreases sharply with an increase in FAD concentration due to inhibition by FAD. More than 90% enzyme activity is lost when FAD concentration is increased from 0.01 to 0.3 mM
iodoacetate
Mn2+
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0.1 mM, about 20% inhibition
p-chloromercuribenzoate
quinacrin
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strong inhibition
Quinine
Sn2+
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0.1 mM, complete inhibition
additional information
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no significant inhibtion at 0.1 mM, Ba2+, Ni2+, Fe2+ and Zn2+. Cysteine (1 mM), 1% inhibition
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
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1 mM, slight stimulation to 103%
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0058 - 0.027
NADH
0.0619
O2
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pH 7.0, 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11000 - 37700
NADH
8
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100.3
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30°C, pH not specified in the publication
130
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pH 7.0, 37°C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 6
pH 4.6: 68% of maximal activity, pH 6.0: 54% of maximal activity
6 - 9.3
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pH 6.0: about 60% of maximal activity, pH 9.3: about 50% of maximal activity
6.5 - 8.5
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higher than 70% of the maximum activity at pH values of 6.5–8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 70
the enzyme is not thermal-sensitive since there is an activity change of only 28% when the temperature varies in the range of 25°C to 70°C
30 - 50
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30°C: about 60% of maximal activity, 50°C: about 65% of maximal activity
30 - 70
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30°C: about 75% of maximal activity, 70°C: about 80% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
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isoelectric focusing
5
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48800
4 * 48800, SDS-PAGE, MALDI mass spectrometry
49919
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x * 49919, calculated from sequence
51000
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1 * 51000, SDS-PAGE
60000
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gel filtration
100000
180000
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gel filtration
196000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals of this protein are grown by sitting-drop variant of the vapour-diffusion method at 20°C in the presence of 34% polyethylene glycol monomethyl ether 2000, 0.1 M sodium acetate and 0.2 M ammonium sulfate at pH 5.4. They belong to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 A, alpha = gamma = 90°, beta = 103.8°. The diffraction limit is 4.0 A
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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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pH 7.0, 1 h, activity markedly decreases
70
half-life: 4.2 h
80
half-life: 2.1 h
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 6 months, enzyme retaines 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
cloned into the T7 promoter-based plasmid pET28a to give pET28a-SpNox and then heterologously overexpressed in Escherichia coli BL21 (DE3)
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expression in Saccharomyces cerevisiae V5
overexpressed in Escherichia coli BL21(DE3)
overexpression in Lactococcus lactis strains, plasmid vector pNZ8020 under the control of the Lactococcus lactis nisA promoter
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
aerobically induced
the expression of noxA is strongly upregulated within 10 min after the growth conditions are altered to a microoxic state
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C44A
-
80% of wild-type activity, production of H2O2 rather than H2O
D292A
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mutant enzyme nearly loses all activity, also loses yellow color, indicating that the amino acid is important for activity
G13A
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mutant enzyme nearly loses all activity, also loses yellow color, indicating that the amino acid is important for activity
G169A
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loses nearly 100% of its activity, indicating that G169 is important for NADH binding
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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the enzyme may prove to be useful for NAD+ regeneration in the production rare L--sugars such as L-ribose, L-ribulose, and L-xylulose