Information on EC 1.14.13.29 - 4-nitrophenol 2-monooxygenase

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

EC NUMBER
COMMENTARY hide
1.14.13.29
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RECOMMENDED NAME
GeneOntology No.
4-nitrophenol 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-nitrophenol + NADH + H+ + O2 = 4-nitrocatechol + NAD+ + H2O
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-nitrophenol degradation II
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Aminobenzoate degradation
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
4-nitrophenol,NADH:oxygen oxidoreductase (2-hydroxylating)
A flavoprotein (FAD).
CAS REGISTRY NUMBER
COMMENTARY hide
91116-87-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
human
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Manually annotated by BRENDA team
male Swiss Webster mice infected by malaria pathogen Plasmodium berghei ANKA
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
New Zealand white male rabbit
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-
Manually annotated by BRENDA team
sheep
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
p-nitrophenol hydroxylase component A; strain DS001 isolated from soil, MTCC 4830
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Manually annotated by BRENDA team
male pigs, surgically castrated pigs, immunocastrated pigs
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
NphA1 oxidizes 4-nitrophenol into 4-nitrocatechol in the presence of FAD, NADH and NphA2 (reduces FAD in the presence of NADH)
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-nitrophenol + NADH + H+ + O2
4-nitrocatechol + NAD+ + H2O
show the reaction diagram
1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer, pH 8.0, slow degradation of substrate
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-
?
3-nitrophenol + NADH + O2
?
show the reaction diagram
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-
-
-
?
4-chlorophenol + NADH + H+ + O2
4-chlorocatechol + NAD+ + H2O
show the reaction diagram
1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer, pH 8.0
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-
?
4-nitrocatechol + NADH + H+ + O2
4-nitrobenzene-1,2,3-triol + NAD+ + H2O
show the reaction diagram
1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer, pH 8.0, very slow degradation of substrate, no unambiguous identification of products by HPLC due to overlaps
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-
?
4-nitrophenol + NAD(P)H + O2
4-nitrocatechol + NAD(P)+ + H2O
show the reaction diagram
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-
-
-
?
4-nitrophenol + NADH + H+ + O2
4-nitrocatechol + NAD+ + H2O
show the reaction diagram
4-nitrophenol + NADH + O2
4-nitrocatechol + NAD+ + H2O
show the reaction diagram
4-nitrophenol + NADPH + O2
4-nitrocatechol + NADP+ + H2O
show the reaction diagram
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-
-
-
?
chlorzoxazone + NADH + O2
?
show the reaction diagram
p-nitrophenol + NADH + H+ + O2
p-nitrocatechol + NAD+ + H2O
show the reaction diagram
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-
-
-
?
p-nitrophenol + NADPH + H+ + O2
p-nitrocatechol + NADP+ + H2O
show the reaction diagram
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200 microM substrate, 0.4 mg microsomal protein, 1 mM NADPH
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?
phenol + NADH + H+ + O2
catechol + NAD+ + H2O
show the reaction diagram
1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer, pH 8.0
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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-nitrophenol + NAD(P)H + O2
4-nitrocatechol + NAD(P)+ + H2O
show the reaction diagram
-
-
-
-
?
4-nitrophenol + NADH + H+ + O2
4-nitrocatechol + NAD+ + H2O
show the reaction diagram
p-nitrophenol + NADH + H+ + O2
p-nitrocatechol + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
p-nitrophenol + NADPH + H+ + O2
p-nitrocatechol + NADP+ + H2O
show the reaction diagram
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200 microM substrate, 0.4 mg microsomal protein, 1 mM NADPH
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
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50% of the activity with NADH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-nitrophenol
alpha-naphthoflavone
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slight
catalase
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30% inhibition at 1000 units
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chlorzoxazone
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mutual competitive inhibition with 4-nitrophenol
Co2+
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slight effect, crude enzyme extract
coumarin
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slight
Cu2+
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94% inhibition at 1 mM,crude enzyme extract
diethyldithiocarbamate
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50% inhibition at 0.002-0.003 mM
FAD
inhibition at concentrations higher than 10 microM FAD, complete inhibition at concentrations higher than 50 microM FAD
Fe2+
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slight effect, crude enzyme extract
Fe3+
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slight effect, crude enzyme extract
Hg2+
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63% inhibition at 1 mM, crude enzyme extract
Horseradish peroxidase
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complete inhibition at 25 units
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mephenytoin
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slight
N-Methylmaleimide
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84% inhibition at 5 mM
Ni2+
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slight effect, crude enzyme extract
p-chloromercuribenzoate
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81% inhibition at 1 mM
quinidine
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slight
sesamin
Sn2+
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57% inhibition at 1 mM,crude enzyme extract
sulfaphenazole
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slight
troleandomycin
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slight
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha-naphthoflavone
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60% activiation at 25 mM
NphR
positive regulatory protein for 4-nitrophenol hydroxylase
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0083 - 0.03
4-nitrocatechol
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.127
4-nitrophenol
Oryctolagus cuniculus
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reconstituted system of purified isozyme 3a, dilaroylglyceryl-3-phosphorylcholine and NADPH-cytochrome P-450 reductase at pH 7.6
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.042
4-nitrophenol
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inhibition of chlorzoxazone 6-hydroxylation
0.047
chlorzoxazone
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inhibition of 4-nitrophenol hydroxylation
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0152 - 0.1943
sesamin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000037
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microsomal fraction
0.000494
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0.00275
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malaria infected mouse, real time kinetic method
0.00411
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mouse without infection, real time kinetic method
11.2
cell extract, 0.3 mM 4-nitrophenol as substrate, 1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer (pH 7.5), 22C
24.5
HiTrap Q-Sepharose purified enzyme, 0.3 mM 4-nitrophenol as substrate, 1 mM NADH, 5 M FAD, 1 mM DTT, NphA1 (approximately 0.5 mg of purified protein), and NphA2 (approximately 0.1 mg of purified protein) in 1 ml of 50 mM Tris-HCl buffer (pH 7.5), 22C
98.3
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entire male pig, pH 6.8
132.8
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surgically castrated pig, pH 6.8
161.5
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immunocastrated pig, pH 6.8
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
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crude enzyme extract
7.4
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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crude enzyme extract
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
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Manually annotated by BRENDA team
additional information
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strain DS001 utilizes methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but cannot grow using hydroquinone as a source of carbon
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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SDS-PAGE
58800
calculated from amino acid sequence
207000
gel filtration chromatography using HPLC, native tetramer
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
4 * x, gel filtration chromatography
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis of crude enzyme extract, complete loss of activity
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Glycerol
more than 80% activity can be kept at least for a week in the presence of 20% glycerol at -20C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 85% loss of activity after 10 days
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0-4C, 30% loss of activity after 7 days, 70% loss of activity after 14 days, crude enzyme extract
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cells centrifuged, washed with TD buffer (50 mM Tris-HCl, 1 mM dithiothreitol (DTT), pH 7.6), centrifuged, bacterial pellet resuspended in same buffer, sonicated, centrifuged, supernatant used as cell extract or further purified with ion-exchange chromatography with a HiTrap Q-Sepharose column, active fractions are desalted with PD-10 desalting column, concentrated with Vivaspin concentrator
liver is homogenized, centrifuged, supernatant mixed with 8 mM calcium chloride, microsomes separated by centrifugation, pellet resuspended in 50 mM Tris-HCl with 0.1 mM ethylendiaminetetraacetic acid and 20% glycerol, pH 7.4
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, phylogenetic analysis
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PCR-amplification, shuttle vector pK4 is used to construct pKPN plasmids containing enzyme gene and reporter gene, hosts are Rhodococcus sp. PN1 and Rhodococcus rhodochrous ATCC 12674 (electroporation), Escherichia coli JM109 is host for propagation and purification of recombinant plasmids
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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malaria decreases the activity of p-nitrophenol-hydroxylase in liver microsomes by 33%, mRNA levels are lowered compared to uninfected mice