BRENDA - Enzyme Database
show all sequences of 1.14.13.29

Mechanism of 4-nitrophenol oxidation in Rhodococcus sp. Strain PN1: characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression

Takeo, M.; Murakami, M.; Niihara, S.; Yamamoto, K.; Nishimura, M.; Kato, D.; Negoro, S.; J. Bacteriol. 190, 7367-7374 (2008)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
additional information
only inducer of nphA1 expression is 4-nitrophenol, no induction of nphA1 gene expression (in the presence of regulator NphR) by phenol, 2-nitrophenol, 3-nitrophenol, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, 4-nitrocatechol
Rhodococcus sp. PN1
NphR
positive regulatory protein for 4-nitrophenol hydroxylase
Rhodococcus sp. PN1
Cloned(Commentary)
Cloned (Commentary)
Organism
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
Rhodococcus sp. PN1
Inhibitors
Inhibitors
Commentary
Organism
Structure
FAD
inhibition at concentrations higher than 10 microM FAD, complete inhibition at concentrations higher than 50 microM FAD
Rhodococcus sp. PN1
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
53000
-
SDS-PAGE
Rhodococcus sp. PN1
58800
-
calculated from amino acid sequence
Rhodococcus sp. PN1
207000
-
gel filtration chromatography using HPLC, native tetramer
Rhodococcus sp. PN1
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
4-nitrophenol + NADH + H+ + O2
Rhodococcus sp. PN1
in the presence of FAD and histidin-tagged NphA2
4-nitrocatechol + NAD+ + H2O
-
-
?
Organic Solvent Stability
Organic Solvent
Commentary
Organism
Glycerol
more than 80% activity can be kept at least for a week in the presence of 20% glycerol at -20°C
Rhodococcus sp. PN1
Organism
Organism
UniProt
Commentary
Textmining
Rhodococcus sp. PN1
Q8RQQ0
-
-
Purification (Commentary)
Purification (Commentary)
Organism
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
Rhodococcus sp. PN1
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
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), 22°C
Rhodococcus sp. PN1
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), 22°C
Rhodococcus sp. PN1
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
3-nitrophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
4-chlorophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-chlorocatechol + NAD+ + H2O
-
-
-
?
4-nitrocatechol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrobenzene-1,2,3-triol + NAD+ + H2O
-
-
-
?
4-nitrophenol + NADH + H+ + O2
in the presence of FAD and histidin-tagged NphA2
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
4-nitrophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
additional information
no degradation of 2-nitrophenol, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate
698597
Rhodococcus sp. PN1
?
-
-
-
-
phenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
catechol + NAD+ + H2O
-
-
-
?
Subunits
Subunits
Commentary
Organism
tetramer
4 * x, gel filtration chromatography
Rhodococcus sp. PN1
Synonyms
Synonyms
Commentary
Organism
NphA1
-
Rhodococcus sp. PN1
two-component 4-nitrophenol hydroxylase
NphA1 (oxidase component) and NphA2 (flavin reductase component)
Rhodococcus sp. PN1
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
-
Rhodococcus sp. PN1
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
-
Rhodococcus sp. PN1
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
additional information
only inducer of nphA1 expression is 4-nitrophenol, no induction of nphA1 gene expression (in the presence of regulator NphR) by phenol, 2-nitrophenol, 3-nitrophenol, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, 4-nitrocatechol
Rhodococcus sp. PN1
NphR
positive regulatory protein for 4-nitrophenol hydroxylase
Rhodococcus sp. PN1
Cloned(Commentary) (protein specific)
Commentary
Organism
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
Rhodococcus sp. PN1
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
-
Rhodococcus sp. PN1
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
FAD
inhibition at concentrations higher than 10 microM FAD, complete inhibition at concentrations higher than 50 microM FAD
Rhodococcus sp. PN1
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
53000
-
SDS-PAGE
Rhodococcus sp. PN1
58800
-
calculated from amino acid sequence
Rhodococcus sp. PN1
207000
-
gel filtration chromatography using HPLC, native tetramer
Rhodococcus sp. PN1
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
4-nitrophenol + NADH + H+ + O2
Rhodococcus sp. PN1
in the presence of FAD and histidin-tagged NphA2
4-nitrocatechol + NAD+ + H2O
-
-
?
Organic Solvent Stability (protein specific)
Organic Solvent
Commentary
Organism
Glycerol
more than 80% activity can be kept at least for a week in the presence of 20% glycerol at -20°C
Rhodococcus sp. PN1
Purification (Commentary) (protein specific)
Commentary
Organism
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
Rhodococcus sp. PN1
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
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), 22°C
Rhodococcus sp. PN1
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), 22°C
Rhodococcus sp. PN1
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
3-nitrophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
4-chlorophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-chlorocatechol + NAD+ + H2O
-
-
-
?
4-nitrocatechol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrobenzene-1,2,3-triol + NAD+ + H2O
-
-
-
?
4-nitrophenol + NADH + H+ + O2
in the presence of FAD and histidin-tagged NphA2
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
4-nitrophenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
4-nitrocatechol + NAD+ + H2O
-
-
-
?
additional information
no degradation of 2-nitrophenol, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate
698597
Rhodococcus sp. PN1
?
-
-
-
-
phenol + NADH + H+ + O2
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
698597
Rhodococcus sp. PN1
catechol + NAD+ + H2O
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
tetramer
4 * x, gel filtration chromatography
Rhodococcus sp. PN1
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
-
Rhodococcus sp. PN1
General Information
General Information
Commentary
Organism
metabolism
NphA1 oxidizes 4-nitrophenol into 4-nitrocatechol in the presence of FAD, NADH and NphA2 (reduces FAD in the presence of NADH)
Rhodococcus sp. PN1
General Information (protein specific)
General Information
Commentary
Organism
metabolism
NphA1 oxidizes 4-nitrophenol into 4-nitrocatechol in the presence of FAD, NADH and NphA2 (reduces FAD in the presence of NADH)
Rhodococcus sp. PN1
Other publictions for EC 1.14.13.29
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
742710
Kallubai
In silico approach to support ...
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Interdiscip. Sci.
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745126
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Interdiscip. Sci. Comput. Life Sci.
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157-167
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-
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3
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8
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4
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3
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7
7
-
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741687
Min
The gene cluster for para-nit ...
Rhodococcus imtechensis, Rhodococcus imtechensis RKJ300
Appl. Environ. Microbiol.
80
6212-6222
2014
-
-
-
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2
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725938
Wagner
In vitro inhibition of 7-ethox ...
Cyprinus carpio, Salmo salar
Mol. Biol. Rep.
40
457-462
2013
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2
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2
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6
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695586
Zamaratskaia
Modulation of porcine cytochro ...
Sus scrofa
Animal
3
1124-1132
2009
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1
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697776
Carvalho
Malaria downmodulates mRNA exp ...
Mus musculus
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616
265-269
2009
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1
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1
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4
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698597
Takeo
Mechanism of 4-nitrophenol oxi ...
Rhodococcus sp. PN1
J. Bacteriol.
190
7367-7374
2008
2
-
1
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1
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3
1
1
5
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1
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7
1
2
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1
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1
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684593
Pakala
Biodegradation of methyl parat ...
Serratia sp. DS001
Appl. Microbiol. Biotechnol.
73
1452-1462
2007
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1
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673929
Vences-Mejia
The effect of aspartame on rat ...
Rattus norvegicus
Hum. Exp. Toxicol.
25
453-459
2006
1
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1
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5
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2
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1
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1
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438907
Hanioka
Effect of alachlor on hepatic ...
Rattus norvegicus
Drug Chem. Toxicol.
25
25-37
2002
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1
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1
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438906
Allis
A kinetic assay for p-nitrophe ...
Rattus norvegicus
Anal. Biochem.
219
49-52
1994
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1
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438908
Tassaneeyakul
Validation of 4-nitrophenol as ...
Homo sapiens
Biochem. Pharmacol.
46
1975-1981
1993
1
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1
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9
2
1
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1
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1
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1
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1
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2
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2
-
-
1
-
1
-
-
-
-
-
9
2
2
1
-
1
-
-
-
-
1
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
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-
438904
Arinc
Lung microsomal p-nitrophenol ...
Ovis aries
Comp. Biochem. Physiol. B
97
455-460
1990
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-
-
-
-
-
-
1
1
-
-
-
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2
-
-
-
-
-
1
1
-
1
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
1
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
1
1
-
1
-
-
-
-
-
1
-
-
-
-
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-
-
-
-
438903
Koop
Hydroxylation of p-nitrophenol ...
Oryctolagus cuniculus
Mol. Pharmacol.
29
399-404
1986
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-
-
-
-
-
1
-
1
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-
-
-
2
-
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1
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-
1
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1
-
-
-
-
-
1
1
-
-
1
-
-
-
-
-
-
1
-
-
-
-
1
-
-
1
-
-
-
-
-
-
1
-
1
-
-
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
438905
Reinke
p-Nitrophenol hydroxylation. A ...
Rattus norvegicus
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13
548-552
1985
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-
-
-
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1
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1
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1
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1
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1
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1
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1
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-
438902
Mitra
A new 4-nitrophenol 2-hydroxyl ...
Nocardia sp.
Biochem. Int.
8
609-615
1984
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-
-
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1
12
1
1
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1
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2
4
-
-
1
-
-
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1
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3
-
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3
-
-
1
-
12
-
1
1
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2
4
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1
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1
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