Information on EC 1.14.13.210 - 4-methyl-5-nitrocatechol 5-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.210
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RECOMMENDED NAME
GeneOntology No.
4-methyl-5-nitrocatechol 5-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-methyl-5-nitrocatechol + NAD(P)H + H+ + O2 = 2-hydroxy-5-methylquinone + nitrite + NAD(P)+ + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,4-dinitrotoluene degradation
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SYSTEMATIC NAME
IUBMB Comments
4-methyl-5-nitrocatechol,NAD(P)H:oxygen 5-oxidoreductase (5-hydroxylating, nitrite-forming)
Contains FAD. The enzyme, isolated from the bacterium Burkholderia sp. DNT, can use both NADH and NADPH, but prefers NADPH. It has a narrow substrate range, but can also act on 4-nitrocatechol.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene dntB
UniProt
Manually annotated by BRENDA team
gene dntB
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-methyl-4-nitrophenol + O2 + NADPH + H+
2-methyl-4-benzoquinone + nitrite + H2O + NADP+
show the reaction diagram
4-methyl-5-nitrocatechol + O2 + NADPH + H+
2-hydroxy-5-methylquinone + H2O + NADP+
show the reaction diagram
4-methyl-5-nitrocatechol + O2 + NADPH + H+
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
show the reaction diagram
4-nitrophenol + O2 + NADPH + H+
4-benzoquinone + nitrite + H2O + NADP+
show the reaction diagram
5-nitrocatechol + O2 + NADPH + H+
2-hydroxyquinone + nitrite + H2O + NADP+
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
4-methyl-5-nitrocatechol + O2 + NADPH + H+
2-hydroxy-5-methylquinone + H2O + NADP+
show the reaction diagram
4-methyl-5-nitrocatechol + O2 + NADPH + H+
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
the enzyme uses either NADH or NADPH as electron donors, and NADPH is the preferred cofactor
NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-nitrophenol
inhibitory at concentrations above 1 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 0.35
4-nitrophenol
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014 - 0.015
4-nitrophenol
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.45
4-nitrophenol
80
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.03
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strain DNT grown on yeast extract, pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzme to homogeneity from strain DNT by anion exchange chromatography and gel filtration
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene dntB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TG1
gene dntB, sequence comparisons
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L380I
site-directed mutagenesis, the mutant shows unaltered activity with 4-nitrophenol compared to the wild-type enzyme
M22L
site-directed mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
M22L/L380I
error-prone PCR, protein engineering for nitrite removal, the mutant enzyme accepts two addtional substrates 4-nitrophenol and 3-methyl-4-nitrophenol. With 4-nitrophenol, the initial rate of the mutant M22L/L380I enzyme is 10fold higher than that of the wild-type enzyme, the catalytic efficiency for 4-nitrophenol degradation is 11fold higher, and with 3-methyl-4-nitrophenol, the initial rate is 4fold higher compared to the wild-type enzyme
M22L/L380M
simultaneous saturation mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
M22S/L380V
simultaneous saturation mutagenesis, the mutant shows 20% enhanced degradation of 4-nitrophenol compared to mutant M22L/L380I
L380I
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site-directed mutagenesis, the mutant shows unaltered activity with 4-nitrophenol compared to the wild-type enzyme
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M22L
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site-directed mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
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M22L/L380I
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error-prone PCR, protein engineering for nitrite removal, the mutant enzyme accepts two addtional substrates 4-nitrophenol and 3-methyl-4-nitrophenol. With 4-nitrophenol, the initial rate of the mutant M22L/L380I enzyme is 10fold higher than that of the wild-type enzyme, the catalytic efficiency for 4-nitrophenol degradation is 11fold higher, and with 3-methyl-4-nitrophenol, the initial rate is 4fold higher compared to the wild-type enzyme
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M22L/L380M
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simultaneous saturation mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
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M22S/L380V
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simultaneous saturation mutagenesis, the mutant shows 20% enhanced degradation of 4-nitrophenol compared to mutant M22L/L380I
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