BRENDA - Enzyme Database

Insights into flavin-based electron bifurcation via the NADH-dependent reduced ferredoxinNADP oxidoreductase structure

Demmer, J.K.; Huang, H.; Wang, S.; Demmer, U.; Thauer, R.K.; Ermler, U.; J. Biol. Chem. 290, 21985-21995 (2015)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.6.1.4
expressed in Escherichia coli C41(DE3) cells
Thermotoga maritima
1.18.1.2
recombinant expression of the NfnAB complex of Thermotoga maritima in Escherichia coli strain under aerobic conditions
Thermotoga maritima
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.6.1.4
sitting drop vapor diffusion method, using 0.225 M NH4H2PO4, 5% (v/v) ethanol, 20% (v/v) glycerol
Thermotoga maritima
1.18.1.2
purified recombinant NfnAB free or in complex with NADH, sitting drop vapour diffusion method, 17.5 mg/ml NfnAB protein in 10 mM MOPS-KOH, pH 7.0, 2 mM DTT, and 0.01 mM FAD, room temperature, for the complex soaking the crystals with 5mM NADH for 40 min, multiple wavelength anomalous dispersion X-ray diffraction structure determinatin and analysis at 2.3-2.4 A resolution, modeling
Thermotoga maritima
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.18.1.2
additional information
-
additional information
kinetic evidence for its bifurcating behavior of the enzyme, cofactor kinetics, overview
Thermotoga maritima
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.18.1.2
cytoplasm
-
Thermotoga maritima
5737
-
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.6.1.4
Fe2+
the enzyme contains an iron-sulfur center, a [4Fe-4S]-center, and a [2Fe-2S]-center
Thermotoga maritima
1.18.1.2
Fe2+
in iron-sulfur clusters
Thermotoga maritima
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
Thermotoga maritima
-
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
?
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
Thermotoga maritima ATCC 43589
-
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
?
1.18.1.2
2 oxidized ferredoxin + NADPH
Thermotoga maritima
-
2 reduced ferredoxin + NADP+ + H+
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.6.1.4
Thermotoga maritima
Q9X1X4
-
-
1.6.1.4
Thermotoga maritima ATCC 43589
Q9X1X4
-
-
1.18.1.2
Thermotoga maritima
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.6.1.4
Strep-Tactin column chromatography and Sephacryl S-200 gel filtration
Thermotoga maritima
1.18.1.2
recombinant NfnAB complex of Thermotoga maritima from Escherichia coli strain under aerobic conditions by ultracentrifugation, and heat treatment for 30 min at 80°C, the supernatant is further purified by affinity chromatography and ultrafiltration. Iron-sulfur cluster reconstitution in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions
Thermotoga maritima
Reaction
EC Number
Reaction
Commentary
Organism
1.18.1.2
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH
mechanism of FAD-based electron bifurcation, overview
Thermotoga maritima
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
-
742880
Thermotoga maritima
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
-
?
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
-
742880
Thermotoga maritima ATCC 43589
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
-
?
1.18.1.2
2 oxidized ferredoxin + NADPH
-
742880
Thermotoga maritima
2 reduced ferredoxin + NADP+ + H+
-
-
-
r
1.18.1.2
2 oxidized ferredoxin + NADPH
complete reversibility of the reaction
742880
Thermotoga maritima
2 reduced ferredoxin + NADP+ + H+
-
-
-
r
1.18.1.2
additional information
the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview. NADH-dependent NADP+ reduction with reduced ferredoxin with a regenerating system composed of Fdox, ferredoxin-dependent [FeFe]-hydrogenase, and 100% H2 as a gas phase. The apparent Km value of NADPH is lower than that of NADH, and that of NADP+ is lower than that of NAD+. Notably, the NADP+-dependent reduction of NAD+ with Fdred is not feasible
742880
Thermotoga maritima
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
Organism
1.6.1.4
heterodimer
1 * 50000 + 1 *30000, SDS-PAGE
Thermotoga maritima
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.6.1.4
FAD
-
Thermotoga maritima
1.18.1.2
FAD
a-FAD and b-FAD, binding of FAD and the iron-sulfur clusters in the NfnAB complex, overview
Thermotoga maritima
1.18.1.2
Ferredoxin
binding site structure, overview
Thermotoga maritima
1.18.1.2
additional information
the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview
Thermotoga maritima
1.18.1.2
NADP+
binding site structure, overview
Thermotoga maritima
1.18.1.2
NADPH
binding site structure, overview
Thermotoga maritima
1.18.1.2
[2Fe-2S] cluster
iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. Binding of FAD and the iron-sulfur clusters in the NfnAB complex
Thermotoga maritima
1.18.1.2
[4Fe-4S] cluster
iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. The proximal [4Fe-4S] cluster is embedded into a rather hydrophilic pocket, and the irons are ligated to three cysteines (Cys51, Cys90, and Cys96) and Glu117. Binding of FAD and the iron-sulfur clusters in the NfnAB complex
Thermotoga maritima
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.6.1.4
expressed in Escherichia coli C41(DE3) cells
Thermotoga maritima
1.18.1.2
recombinant expression of the NfnAB complex of Thermotoga maritima in Escherichia coli strain under aerobic conditions
Thermotoga maritima
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.6.1.4
FAD
-
Thermotoga maritima
1.18.1.2
FAD
a-FAD and b-FAD, binding of FAD and the iron-sulfur clusters in the NfnAB complex, overview
Thermotoga maritima
1.18.1.2
Ferredoxin
binding site structure, overview
Thermotoga maritima
1.18.1.2
additional information
the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview
Thermotoga maritima
1.18.1.2
NADP+
binding site structure, overview
Thermotoga maritima
1.18.1.2
NADPH
binding site structure, overview
Thermotoga maritima
1.18.1.2
[2Fe-2S] cluster
iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. Binding of FAD and the iron-sulfur clusters in the NfnAB complex
Thermotoga maritima
1.18.1.2
[4Fe-4S] cluster
iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. The proximal [4Fe-4S] cluster is embedded into a rather hydrophilic pocket, and the irons are ligated to three cysteines (Cys51, Cys90, and Cys96) and Glu117. Binding of FAD and the iron-sulfur clusters in the NfnAB complex
Thermotoga maritima
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.6.1.4
sitting drop vapor diffusion method, using 0.225 M NH4H2PO4, 5% (v/v) ethanol, 20% (v/v) glycerol
Thermotoga maritima
1.18.1.2
purified recombinant NfnAB free or in complex with NADH, sitting drop vapour diffusion method, 17.5 mg/ml NfnAB protein in 10 mM MOPS-KOH, pH 7.0, 2 mM DTT, and 0.01 mM FAD, room temperature, for the complex soaking the crystals with 5mM NADH for 40 min, multiple wavelength anomalous dispersion X-ray diffraction structure determinatin and analysis at 2.3-2.4 A resolution, modeling
Thermotoga maritima
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.18.1.2
additional information
-
additional information
kinetic evidence for its bifurcating behavior of the enzyme, cofactor kinetics, overview
Thermotoga maritima
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.18.1.2
cytoplasm
-
Thermotoga maritima
5737
-
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.6.1.4
Fe2+
the enzyme contains an iron-sulfur center, a [4Fe-4S]-center, and a [2Fe-2S]-center
Thermotoga maritima
1.18.1.2
Fe2+
in iron-sulfur clusters
Thermotoga maritima
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
Thermotoga maritima
-
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
?
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
Thermotoga maritima ATCC 43589
-
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
?
1.18.1.2
2 oxidized ferredoxin + NADPH
Thermotoga maritima
-
2 reduced ferredoxin + NADP+ + H+
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.6.1.4
Strep-Tactin column chromatography and Sephacryl S-200 gel filtration
Thermotoga maritima
1.18.1.2
recombinant NfnAB complex of Thermotoga maritima from Escherichia coli strain under aerobic conditions by ultracentrifugation, and heat treatment for 30 min at 80°C, the supernatant is further purified by affinity chromatography and ultrafiltration. Iron-sulfur cluster reconstitution in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions
Thermotoga maritima
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
-
742880
Thermotoga maritima
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
-
?
1.6.1.4
NADH + H+ + NADP+ + reduced ferredoxin [iron-sulfur] cluster
-
742880
Thermotoga maritima ATCC 43589
NAD+ + NADPH + oxidized ferredoxin [iron-sulfur] cluster
-
-
-
?
1.18.1.2
2 oxidized ferredoxin + NADPH
-
742880
Thermotoga maritima
2 reduced ferredoxin + NADP+ + H+
-
-
-
r
1.18.1.2
2 oxidized ferredoxin + NADPH
complete reversibility of the reaction
742880
Thermotoga maritima
2 reduced ferredoxin + NADP+ + H+
-
-
-
r
1.18.1.2
additional information
the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview. NADH-dependent NADP+ reduction with reduced ferredoxin with a regenerating system composed of Fdox, ferredoxin-dependent [FeFe]-hydrogenase, and 100% H2 as a gas phase. The apparent Km value of NADPH is lower than that of NADH, and that of NADP+ is lower than that of NAD+. Notably, the NADP+-dependent reduction of NAD+ with Fdred is not feasible
742880
Thermotoga maritima
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.6.1.4
heterodimer
1 * 50000 + 1 *30000, SDS-PAGE
Thermotoga maritima
General Information
EC Number
General Information
Commentary
Organism
1.18.1.2
evolution
NfnA architecturally belongs to the Fnr family, while NfnB is a member of a disulfide oxidoreductase superfamily
Thermotoga maritima
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.18.1.2
evolution
NfnA architecturally belongs to the Fnr family, while NfnB is a member of a disulfide oxidoreductase superfamily
Thermotoga maritima