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Enzyme Nomenclature
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
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EC NUMBER 
COMMENTARY 
1.6.3.3
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RECOMMENDED NAME
GeneOntology No.
NADH oxidase (H2O2-forming)
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
NADH + H+ + O2 = NAD+ + H2O2
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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.
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
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
physiological function
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further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic Thermotoga hypogea from accidental exposure to O2
physiological function
-
although Pyrococcus furiosus is a strict anaerobe, it may tolerate oxygen to some extent. NOX1 may be involved in the response to oxygen at high temperatures
physiological function
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; 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
physiological function
-
Nox-1 is a protective protein against oxygen toxicity
physiological function
the enzyme may be involved in electron transfer reactions resulting in sulfate respiration
physiological function
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further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic Thermotoga hypogea from accidental exposure to O2
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 NADH + H+ + O2
2 NAD+ + 2 H2O
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
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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
-
-
?
NADPH + H+ + O2
NAD+ + H2O2
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
-
-
?
beta-NADH + H+ + O2
beta-NAD+ + H2O2
the enzyme is specific for beta-NADH. No activity with alpha-NADH, alpha-NADPH, or beta-NADPH
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-
?
beta-NADH + H+ + O2
beta-NAD+ + H2O2
the enzyme is specific for beta-NADH. No activity with alpha-NADH, alpha-NADPH, or beta-NADPH
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme also acts as an NADH:ferredoxin oxidoreductase
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme also acts as an NADH:ferredoxin oxidoreductase
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-
?
NADH + H+ + O2
NAD+ + H2O2
the enzyme may be involved in electron transfer reactions resulting in sulfate respiration
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-
?
NADH + H+ + O2
NAD+ + H2O2
the enzyme predominantly produces H2O2. No activity with NADPH. The enzyme also shows activity with 2,6-dichloroindophenol, ferricyanide, menadione, and 2,3-dimethyl-1,4-naphthoquinone
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-
?
NADH + H+ + O2
NAD+ + H2O2
the enzyme predominantly produces H2O2. No activity with NADPH. The enzyme also shows activity with 2,6-dichloroindophenol, ferricyanide, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Very low activity with cytochrome c
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
-
-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH. No formation of H2O
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH. No formation of H2O
-
-
?
NADH + H+ + O2
NAD+ + H2O2
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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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-
?
NADH + H+ + O2
NAD+ + H2O2
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Nox-1 is a protective protein against oxygen toxicity
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-
?
NADH + H+ + O2
NAD+ + H2O2
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Streptococcus mutans NCBI 11723 contains two distinct NADH oxidases, a H2O2-forming and a H2O-forming enzyme
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
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-
?
NADH + H+ + O2
NAD+ + H2O2
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Streptococcus mutans NCBI 11723 contains two distinct NADH oxidases, a H2O2-forming and a H2O-forming enzyme
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
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-
?
NADH + H+ + O2
NAD+ + H2O2
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oxidizes specifically beta-NADH in the presence of molecular oxygen
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-
?
NADH + H+ + O2
NAD+ + H2O2
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oxidizes specifically beta-NADH in the presence of molecular oxygen
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-
?
NADH + H+ + O2
NAD+ + H2O2
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an oxygen-removing system present in Thermotoga maritima is proposed to work in two steps: firstly by converting O2 to hydrogen peroxide by the NADH oxidase, and secondly by reducing the hydrogen peroxide to water by an NADH peroxidase or rubrerythrin or alkyl hydroperoxide reductase
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH. Highest activity using O2 as an electron acceptor. Compared to lower activities for benzyl viologen (20%) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, 7%), while no activity is observed when FAD, FMN, or riboflavin is used as the electron acceptor
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-
?
additional information
?
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the enzyme shows diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c
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-
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additional information
?
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the enzyme shows diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c
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-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NADH + H+ + O2
NAD+ + H2O2
O29852
the enzyme may be involved in electron transfer reactions resulting in sulfate respiration
-
-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
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-
?
NADH + H+ + O2
NAD+ + H2O2
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the enzyme is highly specific for NADH, no activity with NADPH
-
-
?
NADH + H+ + O2
NAD+ + H2O2
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Nox-1 is a protective protein against oxygen toxicity
-
-
?
NADH + H+ + O2
NAD+ + H2O2
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Streptococcus mutans NCBI 11723 contains two distinct NADH oxidases, a H2O2-forming and a H2O-forming enzyme
-
-
?
NADH + H+ + O2
NAD+ + H2O2
-
Streptococcus mutans NCBI 11723 contains two distinct NADH oxidases, a H2O2-forming and a H2O-forming enzyme
-
-
?
NADH + H+ + O2
NAD+ + H2O2
-
an oxygen-removing system present in Thermotoga maritima is proposed to work in two steps: firstly by converting O2 to hydrogen peroxide by the NADH oxidase, and secondly by reducing the hydrogen peroxide to water by an NADH peroxidase or rubrerythrin or alkyl hydroperoxide reductase
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-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
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a flavoprotein, contains 1.8 mol of non-covalently bound FAD per mol of native enzyme
FAD
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flavoprotein. FAD remains enzyme-bound at room temperature. At least 82% of the FAD remains in the enzyme-bound form at 75°C. FMN is not able to substitute for FAD in the substrate-level FAD-dependent portion of the reaction. The Km-value for O2 is above 0.11 mM
FAD
required for activity. 1.1 mol of FAD per mol of enzyme. The FAD cofactor is associated noncovalently with the protein
FAD
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flavoprotein. The enzyme contains 1.9 mol of FAD per mol native enzyme
FAD
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the enzyme is FAD dependent. The activity is 5fold stimulated if the reaction assay contains FAD (0.05 mM). At FAD concentrations above 50 mM the enzyme activity is essentially exogenous flavin independent
FAD
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requires the presence of a flavin cofactor, showing a high specificity for FAD. The enzyme does not contain a flavin molecule
FAD
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requires the presence of a flavin cofactor, showing a high specificity for FAD. The enzyme is purified as an FAD-containing protein
NADH
the NADPH oxidase activity is about 5% compared to the activity with NADH
NADH
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the enzyme is highly specific for NADH, relative activity with NADPH is 2% compared to the activity with NADH
NADH
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the enzyme is highly specific for NADH, little or no activity with NADPH
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
additional information
additional information
divalent cations are not required for activity; divalent cations are not required for activity
additional information
divalent cations are not required for activity; divalent cations are not required for activity
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5,5'-dithiobis-(2-nitrobenzoate)
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1 mM, 7 min, 50% loss of activity
dithiothreitol
2 mM, rapid decrease in activity to less than 10% of the activity
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0037
beta-NADH
pH and temperature not specified in the publication
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
LITERATURE
IMAGE
115
beta-NADH
Pyrococcus horikoshii
O58049
pH and temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.4 - 8.4
pH 4.4: about 70% of maximal activity, pH 8.4: about 90% of maximal activity
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
7.9 - 11
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pH 7.8: about 60% of maximal activity, pH 11.0: about 90% of maximal activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30 - 58
-
the activity increases with the temperature, as tested from 30°C to 58°C
80
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
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 - 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
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
50000
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
70000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
homotetramer
monomer
tetramer
dimer
2 * 48000, the enzyme exists as a dimer and to some extent as a tetramer, SDS-PAGE; 2 * 68000, the enzyme exists as a monomer and to some extent as a dimer, SDS-PAGE
monomer
1 * 68000, the enzyme exists as a monomer and to some extent as a dimer, SDS-PAGE
tetramer
4 * 48000, the enzyme exists as a dimer and to some extent as a tetramer, SDS-PAGE
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 8
-
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
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
70
80
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
90
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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
-
722518
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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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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
LITERATURE
synthesis
synthesis
-
regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L-phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling
synthesis
the enzyme is applicable as a biocatalyst for NAD+ recycling
synthesis
-
regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L-phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling
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synthesis
-
the enzyme is applicable as a biocatalyst for NAD+ recycling
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LINKS TO OTHER DATABASES (specific for EC-Number 1.6.3.3)
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