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Literature summary extracted from

  • Haigler, B.E.; Gibson, D.T.
    Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 (1990), J. Bacteriol., 172, 457-464.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.14.12.12 ferricyanide activation, direct reduction by reductaseNAP in the presence of NADH in vitro Pseudomonas sp.

General Stability

EC Number General Stability Organism
1.14.12.12 purification of reductase leads to significant loss of flavin cofactor Pseudomonas sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.12.12 1,10-phenanthroline 10 mM, 63% inhibition of ferredoxinNAP reductase Pseudomonas sp.
1.14.12.12 4-chloromercuribenzoate 0.0005 mM, 94% inhibition of ferredoxinNAP reductase Pseudomonas sp.
1.14.12.12 iodoacetate 10 mM, 50% inhibition of ferredoxinNAP reductase Pseudomonas sp.
1.14.12.12 N-ethylmaleimide 2 mM, 30% inhibition of ferredoxinNAP reductase Pseudomonas sp.
1.14.12.12 NaN3 40 mM, 46% inhibition of ferredoxinNAP reductase Pseudomonas sp.
1.18.1.3 1,10-phenanthroline 10 mM, 30% inhibition Pseudomonas sp.
1.18.1.3 iodoacetate 10 mM, 50% inhibition Pseudomonas sp.
1.18.1.3 NEM 10 mM, 67% inhibition Pseudomonas sp.
1.18.1.3 PCMB 0.0005 mM, 94% inhibition Pseudomonas sp.
1.18.1.3 Sodium azide 40 mM, 46% inhibition Pseudomonas sp.
1.18.1.7 iodoacetate 50% inhibition at 10 mM Pseudomonas putida
1.18.1.7 N-ethylmaleimide 67% inhibition at 10 mM Pseudomonas putida
1.18.1.7 o-phenanthroline 63% inhibition at 10 mM Pseudomonas putida
1.18.1.7 p-chloromercuribenzoate 94% inhibition at 0.0005 mM Pseudomonas putida
1.18.1.7 Sodium azide 46% inhibition at 40 mM Pseudomonas putida

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.14.12.12 cytoplasm
-
Pseudomonas sp. 5737
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.12.12 Fe2+ component A, i.e. NADH-ferredoxinNAP reductase, of the multienzyme system is an iron-containing flavoprotein containing 1.8 g-atoms Fe2+ and 2 g-atoms sulfur Pseudomonas sp.
1.18.1.3 Iron iron-sulfur flavoprotein, protein contains 1.8 gatom of iron and 2.0 gatom of acid-labile sulfur Pseudomonas sp.
1.18.1.7 Iron the purified protein contains 1.8 g atoms of iron Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.12.12 additional information
-
three component enzyme system consisting of: component A, i.e. ferredoxinNAP reductase, component B, i.e. terminal oxygenase ISPNAP, and component C, i.e. ferredoxinNAP Pseudomonas sp.
1.14.12.12 34900
-
ferredoxinNAP reductase, native PAGE Pseudomonas sp.
1.14.12.12 36000
-
1 * 36000, ferredoxinNAP reductase, SDS-PAGE Pseudomonas sp.
1.14.12.12 37000
-
ferredoxinNAP reductase, gel filtration Pseudomonas sp.
1.14.12.12 37100
-
ferredoxinNAP reductase, deduced from amino acid sequence Pseudomonas sp.
1.18.1.3 34900
-
non-denaturing PAGE Pseudomonas sp.
1.18.1.3 36000
-
1 * 36000, SDS-PAGE Pseudomonas sp.
1.18.1.3 37000
-
gel filtration Pseudomonas sp.
1.18.1.7 36000
-
1 * 36000, SDS-PAGE Pseudomonas putida
1.18.1.7 37000
-
gel filtration Pseudomonas putida
1.18.1.7 37104
-
1 * 37104, calculated from amino acid sequence Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.12.12 naphthalene + NADH + O2 Pseudomonas sp. three-component dioxygenase, uses two proteins containing three redox centers to transfer electrons to the terminal oxygenase (1R,2S)-1,2-dihydronaphthalene-1,2-diol + NAD+
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.14.12.12 Pseudomonas sp.
-
-
-
1.18.1.3 Pseudomonas sp.
-
-
-
1.18.1.3 Pseudomonas sp.
-
NADH-ferredoxinNAP reductase component of naphthalene dioxygenase
-
1.18.1.3 Pseudomonas sp. NCIB 9816
-
-
-
1.18.1.7 Pseudomonas putida Q52126
-
-
1.18.1.7 Pseudomonas putida NCIB 9816 Q52126
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.12.12 ferredoxinNAP reductase, Blue Sepharose, DEAE-cellulose Pseudomonas sp.
1.18.1.3 NADH-ferredoxinNAP reductase component of naphthalene dioxygenase Pseudomonas sp.
1.18.1.7 Sepharose CL-6B column chromatography and DEAE-cellulose column chromatography Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.12.12 naphthalene + NADH + H+ + O2 = (1R,2S)-1,2-dihydronaphthalene-1,2-diol + NAD+ proposed electron transport chain: NADH, ferredoxixinNAP reductase, ferredoxinNAP, terminal oxygenase ISPNAP Pseudomonas sp.

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.12.12 397
-
cytochrome c reduction by ferredoxinNAP reductase Pseudomonas sp.
1.18.1.3 397
-
-
Pseudomonas sp.
1.18.1.7 397
-
purified enzyme, pH and temperature not specified in the publication Pseudomonas putida

Storage Stability

EC Number Storage Stability Organism
1.14.12.12 -20°C, ferredoxinNAP reductase, 1 month, minimal loss of activity, prolonged storage leads to precipitation when preparation is heated above 5°C Pseudomonas sp.
1.14.12.12 0-5°C, ferredoxinNAP reductase, 5 days, 30% loss of activity Pseudomonas sp.
1.18.1.3 -20°C, 1 month, minimal loss of activity Pseudomonas sp.
1.18.1.3 0-5°C, 30% loss of activity after 5 days Pseudomonas sp.
1.18.1.7 -20°C, purified enzyme, 1 month, minimal loss of activity Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.12.12 naphthalene + NADH + O2 3 component enzyme system consisting of ferredoxinNAP reductase, ferredoxinNAP and oxygenase ISPNAP, ferredoxinNAP reductase reduces: 2,6-dichlorophenolindophenol, ferricyanide, nitro blue tetrazolium and cytochrome c, in the presence of FAD ferredoxinNAP reductase transfers electrons to ferredoxin Pseudomonas sp. cis-(1R,2S)-1,2-dihydronaphthalene-1,2-diol + NAD+
-
?
1.14.12.12 naphthalene + NADH + O2 three-component dioxygenase, uses two proteins containing three redox centers to transfer electrons to the terminal oxygenase Pseudomonas sp. (1R,2S)-1,2-dihydronaphthalene-1,2-diol + NAD+
-
?
1.18.1.3 2 ferricyanide + NADH
-
Pseudomonas sp. 2 ferrocyanide + NAD+ + H+
-
?
1.18.1.3 2 ferricyanide + NADH
-
Pseudomonas sp. NCIB 9816 2 ferrocyanide + NAD+ + H+
-
?
1.18.1.3 2 ferricytochrome c + NADH
-
Pseudomonas sp. 2 ferrocytochrome c + NAD+ + H+
-
?
1.18.1.3 2 ferricytochrome c + NADH
-
Pseudomonas sp. NCIB 9816 2 ferrocytochrome c + NAD+ + H+
-
?
1.18.1.3 NADH + oxidized 2,6-dichlorophenolindophenol + H+
-
Pseudomonas sp. NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
1.18.1.3 NADH + oxidized 2,6-dichlorophenolindophenol + H+
-
Pseudomonas sp. NCIB 9816 NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
1.18.1.3 NADH + oxidized nitro blue tetrazolium + H+
-
Pseudomonas sp. NAD+ + reduced nitro blue tetrazolium
-
?
1.18.1.3 NADH + oxidized nitro blue tetrazolium + H+
-
Pseudomonas sp. NCIB 9816 NAD+ + reduced nitro blue tetrazolium
-
?
1.18.1.3 NADPH + oxidized cytochrome c 39% of the activity with NADH Pseudomonas sp. NADP+ + reduced cytochrome c
-
?
1.18.1.3 NADPH + oxidized cytochrome c 39% of the activity with NADH Pseudomonas sp. NCIB 9816 NADP+ + reduced cytochrome c
-
?
1.18.1.7 2 ferricyanide + NADH best substrate Pseudomonas putida 2 ferrocyanide + NAD+ + H+
-
?
1.18.1.7 2 ferricyanide + NADH best substrate Pseudomonas putida NCIB 9816 2 ferrocyanide + NAD+ + H+
-
?
1.18.1.7 2 ferricytochrome c + NADH
-
Pseudomonas putida 2 ferrocytochrome c + NAD+ + H+
-
?
1.18.1.7 2 ferricytochrome c + NADH
-
Pseudomonas putida NCIB 9816 2 ferrocytochrome c + NAD+ + H+
-
?
1.18.1.7 NADH + H+ + oxidized 2,6-dichlorophenolindophenol
-
Pseudomonas putida NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
1.18.1.7 NADH + H+ + oxidized 2,6-dichlorophenolindophenol
-
Pseudomonas putida NCIB 9816 NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
1.18.1.7 oxidized cytochrome c + NADPH NADPH is also effective as an electron donor but yields only 39% of the activity obtained with NADH in the cytochrome c reductase assay Pseudomonas putida reduced cytochrome c + NADP+ + H+
-
?
1.18.1.7 oxidized cytochrome c + NADPH NADPH is also effective as an electron donor but yields only 39% of the activity obtained with NADH in the cytochrome c reductase assay Pseudomonas putida NCIB 9816 reduced cytochrome c + NADP+ + H+
-
?
1.18.1.7 oxidized Nitro Blue tetrazolium + NADH
-
Pseudomonas putida reduced Nitro Blue tetrazolium + NAD+ + H+
-
?
1.18.1.7 oxidized Nitro Blue tetrazolium + NADH
-
Pseudomonas putida NCIB 9816 reduced Nitro Blue tetrazolium + NAD+ + H+
-
?

Subunits

EC Number Subunits Comment Organism
1.14.12.12 monomer 1 * 36000, ferredoxinNAP reductase, SDS-PAGE Pseudomonas sp.
1.18.1.3 monomer 1 * 36000, SDS-PAGE Pseudomonas sp.
1.18.1.7 monomer 1 * 36000, SDS-PAGE Pseudomonas putida
1.18.1.7 monomer 1 * 37104, calculated from amino acid sequence Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.18.1.3 NADH-ferredoxinNAP reductase component of naphthalene dioxygenase multicomponent enzyme system Pseudomonas sp.
1.18.1.7 NADH-ferredoxinNAP reductase
-
Pseudomonas putida

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.12.12 25
-
room temperature, ferredoxinNAP reductase, t1/2: 8 h Pseudomonas sp.
1.18.1.3 0.5
-
5 d, 30% loss of activity Pseudomonas sp.
1.18.1.3 21
-
room temperature, 8 h, 50% loss of activity Pseudomonas sp.
1.18.1.7
-
20 the purified enzyme is unstable at room temperature. It loses 50% of its cytochrome c reductase activity within the first 8 h. At 0 to 5°C, the purified enzyme retains 70% of its cytochrome c reductase activity after 5 days Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.12.12 additional information
-
ferredoxin reductaseNAP, pI: 6.3 Pseudomonas sp.

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.12.12 FAD 1 mol FAD/mol enzyme in flavin-reconstituted protein Pseudomonas sp.
1.14.12.12 FAD component A, ferredoxinNAP reductase is a flavoprotein, enzyme can use both FAD and FMN but exhibits slightly higher affinity for FAD Pseudomonas sp.
1.14.12.12 FAD addition enhances ferredoxinNAP reductase activity with all in vitro electron-acceptors, e.g. cytochrome c, 2,6-dichlorophenolindophenol, Nitroblue tetrazolium and ferricyanide Pseudomonas sp.
1.14.12.12 FMN requirement, ferredoxinNAP reductase is a flavoprotein, enzyme can use both FAD and FMN but exhibits slightly higher affinity for FAD, addition stimulates dioxygenase activity by 53% of FAD-stimulation Pseudomonas sp.
1.14.12.12 NADH
-
Pseudomonas sp.
1.14.12.12 NADPH ferredoxinNAP reductase Pseudomonas sp.
1.14.12.12 NADPH can replace NADH with 39% Pseudomonas sp.
1.18.1.3 FAD 1 mol of FAD is bound per mol of enzyme Pseudomonas sp.
1.18.1.3 NADH
-
Pseudomonas sp.
1.18.1.3 NADPH 39% of the activity with NADH in the reaction with cytochrome c Pseudomonas sp.
1.18.1.7 FAD the enzyme binds 1 mol of FAD per mol of enzyme protein Pseudomonas putida
1.18.1.7 NADH
-
Pseudomonas putida

pI Value

EC Number Organism Comment pI Value Maximum pI Value
1.18.1.7 Pseudomonas putida calculated from amino acid sequence
-
6.3

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
1.18.1.7 10
-
pH and temperature not specified in the publication Pseudomonas putida iodoacetate