Information on EC 1.7.3.1 - nitroalkane oxidase

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

EC NUMBER
COMMENTARY hide
1.7.3.1
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RECOMMENDED NAME
GeneOntology No.
nitroalkane oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a nitroalkane + H2O + O2 = an aldehyde or ketone + nitrite + H2O2
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
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-
-
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reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitroethane degradation
-
-
Nitrogen metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
nitroalkane:oxygen oxidoreductase
Has an absolute requirement for FAD [4]. While nitroethane may be the physiological substrate [2], the enzyme also acts on several other nitroalkanes, including 1-nitropropane, 2-nitropropane, 1-nitrobutane, 1-nitropentane, 1-nitrohexane, nitrocyclohexane and some nitroalkanols [4]. Differs from EC 1.13.11.16, nitronate monooxygenase, in that the preferred substrates are neutral nitroalkanes rather than anionic nitronates [4].
CAS REGISTRY NUMBER
COMMENTARY hide
9029-36-1
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-nitroalkane + O2 + H2O
aldehyde + H2O2 + nitrite
show the reaction diagram
-
-
-
-
?
1-nitrobutane + O2
butyraldehyde + HNO2
show the reaction diagram
-
-
-
-
?
1-nitrobutane + O2 + H2O
butyraldehyde + nitrite + H2O2
show the reaction diagram
1-nitrohexane + O2
hexanaldehyde + HNO2
show the reaction diagram
-
-
-
-
?
1-nitrohexane + O2 + H2O
hexanal + nitrite + H2O2
show the reaction diagram
1-nitrohexane + O2 + H2O
hexanaldehyde + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
1-nitrooctane + O2 + H2O
octanal + nitrite + H2O2
show the reaction diagram
-
-
-
?
1-nitropentane + O2
pentanaldehyde + HNO2
show the reaction diagram
-
-
-
-
?
1-nitropropane + H2O
propanal + nitrite + H2O2
show the reaction diagram
-
-
-
?
1-nitropropane + H2O + O2
propionaldehyde + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
1-nitropropane + O2
propionaldehyde + HNO2
show the reaction diagram
-
-
-
-
?
2-nitropropane + H2O
propanone + nitrite + H2O2
show the reaction diagram
-
-
-
?
2-nitropropane + H2O + O2
acetone + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
2-nitropropane + O2
acetone + HNO2
show the reaction diagram
-
-
-
-
?
2-nitropropane + O2
acetone + nitrite
show the reaction diagram
-
-
-
-
?
3-nitropropanoic acid + H2O + O2
2-oxopropanoic acid + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
nitroalkane + O2 + H2O
aldehyde or ketone + nitrite + H2O2
show the reaction diagram
-
neutral nitroalkanes
-
-
?
nitroethane + H2O
ethanal + nitrite + H2O2
show the reaction diagram
nitroethane + H2O + O2
?
show the reaction diagram
-
-
-
-
?
nitroethane + H2O + O2
acetaldehyde + nitrite + H2O2
show the reaction diagram
nitroethane + O2
acetaldehyde + HNO2
show the reaction diagram
nitroethane + O2 + H2O
acetaldehyde + nitrite + H2O2
show the reaction diagram
-
-
-
?
nitroethane + O2 + H2O
ethanal + nitrite + H2O2
show the reaction diagram
nitroethane + O2 + H2O
ethanal + nitrite + H2O2 + H+
show the reaction diagram
phenylnitromethane + O2
benzaldehyde + HNO2
show the reaction diagram
-
-
-
-
?
phenylnitromethane + O2 + H2O
? + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
phenylnitromethane + O2 + H2O
phenylaldehyde + nitrite + H2O2
show the reaction diagram
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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
nitroethane + H2O + O2
acetaldehyde + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
nitroethane + O2 + H2O
ethanal + nitrite + H2O2
show the reaction diagram
-
-
-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
probably not a hemoprotein
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-nitrohexane
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-
1-nitrooctane
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acetaldehyde
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noncompetitive inhibitor versus nitroethane due to formation of a dead-end complex between the oxidized enzyme and the product, competitive inhibitor versus oxygen
Benzoate
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competitive
Boric acid
-
-
Butyraldehyde
-
noncompetitive inhibitor versus nitroethane due to formation of a dead-end complex between the oxidized enzyme and the product
cyanide
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inactivation of the enzyme by trapping of the electrophilic imine reaction intermediate, formation of a cyanoalkyl intermediate, reaction with the substrate after proton abstraction but before flavin oxidation
Maleic acid
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-
NaCl
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slight
NaCN
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slight
NH2OH
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slight
nitroethane
pentanoate
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phenylacetic acid
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competitive
Sodium cyanide
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2 mM and above, complete inhibition
spermine
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weak inhibitor at pH 8.0
valerate
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competitive
additional information
-
no substrate inhibition
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
imidazole
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unprotonated imidazole causes a 5fold increase in maximal velocity
additional information
-
enzyme induction by growth on nitroethane
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03 - 0.18
1-nitrobutane
0.04 - 0.2
1-nitrohexane
0.03 - 0.3
1-nitrooctane
35.7
1-Nitropropane
pH 8.0, 28C
83.5
2-Nitropropane
pH 8.0, 28C
5.1
Benzoate
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-
0.042 - 26.8
nitroethane
0.011 - 0.39
O2
13.1
phenylacetic acid
-
-
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3 - 2
1-nitrohexane
0.2 - 4.4
1-nitrooctane
0.037 - 15
nitroethane
2.6 - 15
O2
additional information
additional information
Fusarium oxysporum
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-
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004 - 6.3
nitroethane
1093
38 - 580
O2
9
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
130
1-nitrohexane
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mutant S276A, pH 8.0, 30C
8.3 - 26
1-nitrooctane
2 - 152
nitroethane
200
spermine
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at pH 8.0
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 7.6
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half-maximal activity at pH 6.2 and 7.6
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 27
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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temperature-dependency analysis
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36700
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x * 36700, calculated
37000
x * 37000, calculated
47955
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2 * 47955, MALDI-TOF mass spectrometry; 4 * 47955, MALDI-TOF mass spectrometry
48162
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x * 48162, amino acid sequence calculation, enzyme forms homodimers and homotetramers with a preference for the latter
147300
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equilibrium sedimentation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
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x-ray crystallography
oligomer
tetramer
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4 * 47955, MALDI-TOF mass spectrometry
additional information
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enzyme structure analysis, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallization data of mutant D402N in complex with 1-nitrohexane or 1-nitrooctane show the presence of substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. The strucuture of wild-type enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Data for mutant S276A in complex with nitrohexane
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hanging drop vapour diffusion method
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hexagonal rod-shaped crystals of R409K and D402E NAO were obtained using hanging drop vapor-diffusion methods
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purified recombinant wild-type enzyme, crystallization of the native enzyme in 2 different crystal forms and of the selenomethionine-labeled enzyme in a third one, hanging drop vapour diffusion method, sodium cacodylate buffer, pH 7.5, containing spermidine hydrochloride, and PEG 4000 at varying concentrations for all 3 mixtures, crystal form 2 requires addition of 1,6-hexanediol at 8% w/v, crystal form 3 requires DTT at 10 mM, 4C, 10-14 days, X-ray diffraction structure determinations and analysis at 3.2-2.0 A resolution or below, three-wavelength MAD data
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vapor diffusion method, using 2.5 M magnesium sulfate, 0.1 M 2-(N-morpholino) ethanesulfonic acid buffer, pH 6.0, and 18% (v/v) glycerol
native and a selenomethionine-substituted enzyme, to 1.9 A resolution. Primitive orthorhombic space group P21, with unit-cell parameters a = 70.06, b = 55.43, c = 87.74 A, beta = 96.56 for native NAO and a = 69.89, b = 54.83, c = 88.20 A, beta = 95.79 for selenomethionine-substituted enzyme
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wild-type and mutant H179D. Enzyme consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a alpha-alpha-alpha-beta-alpha-beta-alpha fold. It shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
-
1 h, 0C, complete loss of activity
395013
11
-
1 h, 0C, full activity recovered after readjusting the solution to pH 7.0
395013
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
-
10 s, 70% loss of activity
100
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2 min, complete loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis: stable to
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, weeks, slow decrease of activity
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in refrigerator for several days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography
recombinant enzyme
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recombinant enzyme from Escherichia coli
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recombinant wild-type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
expression in Escherichia coli strains BL21(DE3) and methionine auxotroph B834(DE3)
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expression of wild-type and mutant enzymes
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recombinant expression of wild-type and mutant D402E enzymes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C397S
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the mutation results in decreases in the rate constants for removal of the substrate proton by about 5fold and decreases in the rate constant for product release of about 2fold, the mutant enzyme is less stable than the wild type enzyme
D402A
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site-directed mutation of the active site base, 20fold reduced catalytic efficiency with neutral nitroethane as substrate compared to the wild-type enzyme, while the wild-type enzyme prefers the neutral substrate the mutant prefers the anion substrate form, altered pH-dependence with both substrate forms compared to the wild-type enzyme
R409K
-
the mutation results in a decrease in the rate constant for proton abstraction of 100fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409; the mutation results in a decrease in the rate constant for proton abstraction of 100fold. Analysis of the three-dimensional structure of the R409K enzyme shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409
S171A
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the mutation results in decreases in the rate constants for removal of the substrate proton by about 5fold and decreases in the rate constant for product release of about 2fold
S171T
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the mutation results in decreases in the rate constants for removal of the substrate proton by about 5fold and decreases in the rate constant for product release of about 2fold, the mutation alters the rate constant for flavin oxidation
S171V
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the mutation results in decreases in the rate constants for removal of the substrate proton by about 5fold and decreases in the rate constant for product release of about 2fold, the mutation alters the rate constant for flavin oxidation
S276A
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more than 20fold decrease in catalytic efficiency
Y398F
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the mutation results in decreases in the rate constants for removal of the substrate proton by about 5fold and decreases in the rate constant for product release of about 2fold, the mutant enzyme is less stable than the wild type enzyme
D399N
the mutation decreases the kcat/KM value for nitroethane over 2 orders of magnitude
R406K
the mutation decreases the kcat/KM value for nitroethane about 64fold
S373A
the mutation decreases the kcat/KM value for nitroethane about 3fold
H179D
residue is spatially adjacent to FMN, mutation results in the loss of enzyme activity
H179K
residue is spatially adjacent to FMN, mutation results in the loss of enzyme activity
H179V
residue is spatially adjacent to FMN, mutation results in the loss of enzyme activity
additional information
naoA disruption accelerates growth of the naoA-disruption mutant, which can restore its phenotype and morphology as a wild-type strain by complementation of a single copy number of naoA inserted into the chromosome. The introduction of an extra copy of naoA into the wild-type strain results in delayed growth