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Literature summary extracted from

  • Takeo, M.; Murakami, M.; Niihara, S.; Yamamoto, K.; Nishimura, M.; Kato, D.; Negoro, S.
    Mechanism of 4-nitrophenol oxidation in Rhodococcus sp. Strain PN1: characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression (2008), J. Bacteriol., 190, 7367-7374.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.14.13.29 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
1.14.13.29 NphR positive regulatory protein for 4-nitrophenol hydroxylase Rhodococcus sp. PN1

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.29 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

EC Number Inhibitors Comment Organism Structure
1.14.13.29 FAD inhibition at concentrations higher than 10 microM FAD, complete inhibition at concentrations higher than 50 microM FAD Rhodococcus sp. PN1

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.13.29 53000
-
 SDS-PAGE Rhodococcus sp. PN1
1.14.13.29 58800
-
 calculated from amino acid sequence Rhodococcus sp. PN1
1.14.13.29 207000
-
 gel filtration chromatography using HPLC, native tetramer Rhodococcus sp. PN1

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.29 4-nitrophenol + NADH + H+ + O2 Rhodococcus sp. PN1 in the presence of FAD and histidin-tagged NphA2 4-nitrocatechol + NAD+ + H2O
-
 ?

Organic Solvent Stability

EC Number Organic Solvent Comment Organism
1.14.13.29 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

EC Number Organism UniProt Comment Textmining
1.14.13.29 Rhodococcus sp. PN1 Q8RQQ0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.29 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.29 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
1.14.13.29 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.29 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 Rhodococcus sp. PN1 4-nitrocatechol + NAD+ + H2O
-
 ?
1.14.13.29 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 Rhodococcus sp. PN1 4-chlorocatechol + NAD+ + H2O
-
 ?
1.14.13.29 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 Rhodococcus sp. PN1 4-nitrobenzene-1,2,3-triol + NAD+ + H2O
-
 ?
1.14.13.29 4-nitrophenol + NADH + H+ + O2 in the presence of FAD and histidin-tagged NphA2 Rhodococcus sp. PN1 4-nitrocatechol + NAD+ + H2O
-
 ?
1.14.13.29 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 Rhodococcus sp. PN1 4-nitrocatechol + NAD+ + H2O
-
 ?
1.14.13.29 additional information no degradation of 2-nitrophenol, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate Rhodococcus sp. PN1 ?
-
 ?
1.14.13.29 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 Rhodococcus sp. PN1 catechol + NAD+ + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.29 tetramer 4 * x, gel filtration chromatography Rhodococcus sp. PN1

Synonyms

EC Number Synonyms Comment Organism
1.14.13.29 NphA1
-
 Rhodococcus sp. PN1
1.14.13.29 two-component 4-nitrophenol hydroxylase NphA1 (oxidase component) and NphA2 (flavin reductase component) Rhodococcus sp. PN1

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.29 8
-
-
 Rhodococcus sp. PN1

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.29 FAD
-
 Rhodococcus sp. PN1

General Information

EC Number General Information Comment Organism
1.14.13.29 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