Information on EC 1.14.12.24 - 2,4-dinitrotoluene dioxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.12.24
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RECOMMENDED NAME
GeneOntology No.
2,4-dinitrotoluene dioxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2,4-dinitrotoluene + NADH + O2 = 4-methyl-5-nitrocatechol + nitrite + NAD+
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,4-dinitrotoluene degradation
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Microbial metabolism in diverse environments
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Nitrotoluene degradation
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SYSTEMATIC NAME
IUBMB Comments
2,4-dinitrotoluene,NADH:oxygen oxidoreductase (4,5-hydroxylating, nitrite-releasing)
This enzyme, characterized from the bacterium Burkholderia sp. strain DNT, is a member of the naphthalene family of bacterial Rieske non-heme iron dioxygenases. It comprises a multicomponent system, containing a Rieske [2Fe-2S] ferredoxin, an NADH-dependent flavoprotein reductase (EC 1.18.1.3, ferredoxin---NAD+ reductase), and an alpha3beta3 oxygenase. The enzyme forms a cis-dihydroxylated product that spontaneously rearranges to form a catechol with accompanying release of nitrite. It does not act on nitrobenzene. cf. EC 1.14.12.23, nitroarene dioxygenase.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2,4-benzenetriol + NADH + O2
?
show the reaction diagram
2,4-dinitrotoluene + NADH + H+ + O2
4-methyl-5-nitrocatechol + nitrite + NAD+
show the reaction diagram
2,4-dinitrotoluene + NADH + O2
4-methyl-5-nitrocatechol + nitrite + NAD+
show the reaction diagram
2,6-dinitrotoluene + NADH + O2
3-methyl-4-nitrocatechol + nitrite + NAD+
show the reaction diagram
2-amino-3-nitrotoluene + NADH + H+ + O2
2-amino-3-nitrobenzylalcohol + NAD+ + H2O
show the reaction diagram
2-amino-5-nitrotoluene + NADH + H+ + O2
2-amino-5-nitrobenzyl alcohol + aminonitrocresol + NAD+ + H2O
show the reaction diagram
2-amino-6-nitrotoluene + NADH + H+ + O2
2-amino-4-nitro-3-cresol + 3-amino-5-nitro-4-cresol + NAD+ + H2O
show the reaction diagram
2-methoxyphenol + NADH + O2
methoxyhydroquinone + NAD+ + H2O
show the reaction diagram
2-nitrophenol + NADH + O2
nitrohydroquinone + 3-nitrocatechol + NAD+ + H2O
show the reaction diagram
2-nitrotoluene + NADH + H+ + O2
2-nitrobenzyl alcohol + NAD+ + ?
show the reaction diagram
2-nitrotoluene + NADH + H+ + O2
2-nitrobenzyl alcohol + NAD+ + H2O
show the reaction diagram
2-nitrotoluene + NADH + O2
?
show the reaction diagram
3-amino-4-nitrotoluene + NADH + H+ + O2
3-amino-4-nitrobenzyl alcohol + aminonitrocresol + NAD+ + H2O
show the reaction diagram
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?
3-methyl-4-nitrophenol + NADH + O2
?
show the reaction diagram
3-nitrophenol + NADH + O2
4-nitrocatechol + NAD+ + H2O
show the reaction diagram
3-nitrotoluene + NADH + H+ + O2
3-nitrobenzyl alcohol + NAD+ + ?
show the reaction diagram
4-amino-2-nitrotoluene + NADH + H+ + O2
aminonitrocresol + NAD+ + H2O
show the reaction diagram
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?
4-amino-3-nitrotoluene + NADH + O2
4-amino-3-nitrobenzylalcohol + aminonitrocresol + ?
show the reaction diagram
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?
4-methyl-5-nitrocatechol + nitrite + NAD+
2,4-dinitrotoluene + NADH + O2
show the reaction diagram
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r
4-nitrocatechol + NADH + O2
?
show the reaction diagram
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low activity
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?
4-nitrotoluene + NADH + H+ + O2
4-nitrobenzyl alcohol + NAD+ + ?
show the reaction diagram
4-nitrotoluene + NADH + O2
4-methylcatechol + nitrite + NAD+
show the reaction diagram
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?
5-amino-2-nitrotoluene + NADH + H+ + O2
2-amino-5-nitro-o-cresol + NAD+ + H2O
show the reaction diagram
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?
acenaphthene + NADH + H+ + O2
1-acenaphthenol + NAD+ + ?
show the reaction diagram
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?
indan + NADH + H+ + O2
1-indanol + 1-indanone + indene + NAD+
show the reaction diagram
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?
indene + NADH + H+ + O2
1-indenol + 1,2-dihydroxyindan + NAD+
show the reaction diagram
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?
indole + NADH + H+ + O2
indigo + NAD+ + ?
show the reaction diagram
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?
indoline + NADH + H+ + O2
indole + NAD+ + ?
show the reaction diagram
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?
naphthalene + NADH + H+ + O2
(+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene + NAD+
show the reaction diagram
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?
naphthalene + NADH + H+ + O2
(+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene + NAD+ + ?
show the reaction diagram
naphthalene + NADH + O2
(1R,3S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene + NAD+ + H2O
show the reaction diagram
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?
phenetole + NADH + H+ + O2
phenol + NAD+ + ?
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
2,4-dinitrotoluene + NADH + H+ + O2
4-methyl-5-nitrocatechol + nitrite + NAD+
show the reaction diagram
2,4-dinitrotoluene + NADH + O2
4-methyl-5-nitrocatechol + nitrite + NAD+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD+
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2fold enhancement of activity in the presence of NAD+
NADP+
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2fold enhancement of activity in the presence of NADP+
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
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required for activity, 238.5% activity at 1 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
deoxycholate
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about 68% residual activity at 6% (v/v)
Dodecyl sulfate
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about 40% residual activity at 1% (v/v)
spermidine
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80% residual activity at 0.05 mg
Triton X-100
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about 50% residual activity at 2% (v/v)
Tween 20
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about 36% residual activity at 2% (v/v)
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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required for maximal activity, 292.3% activity at 1% (v/v)
ATP
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124% activity at 0.1 mM
FMN
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230.7% activity at 0.1 mM
GTP
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116% activity at 0.1 mM
lipase
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176% activity at 1 mg per g wet weight of cells
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NADP+
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2fold enhancement of activity in the presence of NADP+
phosphatidylcholine
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125% activity at 0.05 mg
phosphatidylethanolamine
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144% activity at 0.1 mg per g wet weight of cells
Phospholipase A
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125% activity at 0.01 mg
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Vitreoscilla hemoglobin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.18
2,4-dinitrotoluene
0.05
O2
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at pH 7.0 and 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.38
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with naphthalene as substrate, pH and temperature not specified in the publication
2.4
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crude extract, at pH 7.0 and 37C
19
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crude enzyme, at pH 7.2 and 37C
37.3
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after 15.7fold, at pH 7.0 and 37C
385
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after 20.3fold purification, at pH 7.2 and 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
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the enzyme is stable when incubated at pH 6 and 7, but its optimal activity is at pH 7.0. The activity is decreased significantly when incubated at pH 4.0, 5.0, 8.0, and 10.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 45
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activity rapidly diminishes when the enzyme is heated above 45C. At 20C, the activity is decreased about 25% compared with 37C. At 15C, the activity decreases about 85% compared with 37C, and at 4-5C, the activity is totally absent
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
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x * 50000, SDS-PAGE
100000
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gel filtration
171000
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calculated from amino acid sequence
187000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotrimer
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 4
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the enzyme shows 5% to 15% loss in catalytic activity when left at 0-4C for several weeks
25 - 37
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the enzyme is stable for 2 days when incubated with substrate at 37C, 30C, and 25C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, column chromatography, and Sephadex G-100 gel filtration
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DEAE cellulose column chromatography and Sephadex G150 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli JM109 cells
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expressed in Escherichia coli JM109(DE3) cells
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expressed in Escherichia coli JVQ2 cells
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expressed in Escherichia coli PFJS39 cells
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expressed in Escherichia coli TG1 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V350F
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the mutant of the alpha subunit is inactive with 2,4-dinitrotoluene but shows significantly increased activity towards 2-nitrophenol (47times), 3-nitrophenol (34times), and 2-methoxyphenol (174times) as well as an expanded substrate range that now includes 3-methoxyphenol, o-cresol, and m-cresol (wild type enzyme has no detectable activity for these substrates). The mutant also exhibits 10fold enhanced activity towards naphthalene forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene
V350M
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the mutant is inactive with 2,4-dinitrotoluene but displays increased activity towards 2-nitrophenol (20times) and 2-methoxyphenol (162times) as well as novel activity towards 2-cresol compared to the wild type enzyme. The mutant also exhibits 10fold enhanced activity towards naphthalene forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene
V350F
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the mutant of the alpha subunit is inactive with 2,4-dinitrotoluene but shows significantly increased activity towards 2-nitrophenol (47times), 3-nitrophenol (34times), and 2-methoxyphenol (174times) as well as an expanded substrate range that now includes 3-methoxyphenol, o-cresol, and m-cresol (wild type enzyme has no detectable activity for these substrates). The mutant also exhibits 10fold enhanced activity towards naphthalene forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene
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V350M
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the mutant is inactive with 2,4-dinitrotoluene but displays increased activity towards 2-nitrophenol (20times) and 2-methoxyphenol (162times) as well as novel activity towards 2-cresol compared to the wild type enzyme. The mutant also exhibits 10fold enhanced activity towards naphthalene forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene
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I204L
the mutant transforms both 2,6-dinitrotoluene and 2,4-dinitrotoluene 2fold faster than the wild type enzyme and exhibits activity with 2,5- and 2,3-dinitrotoluene
I204L
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the mutant transforms both 2,6-dinitrotoluene and 2,4-dinitrotoluene 2fold faster than the wild type enzyme and exhibits activity with 2,5- and 2,3-dinitrotoluene
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I204Y
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the mutant transforms both 2,6-dinitrotoluene 2,5fold faster than the wild type enzyme and exhibits activity with 2,5- and 2,3-dinitrotoluene; the mutation results in 2-4fold faster oxidization of the aminonitrotoluenes compared to the wild type enzyme
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