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Literature summary extracted from

  • Demmer, J.K.; Huang, H.; Wang, S.; Demmer, U.; Thauer, R.K.; Ermler, U.
    Insights into flavin-based electron bifurcation via the NADH-dependent reduced ferredoxin NADP oxidoreductase structure (2015), J. Biol. Chem., 290, 21985-21995 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) 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 (Comment) 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 Comment 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 Comment Organism GeneOntology No. Textmining
1.18.1.2 cytoplasm
-
Thermotoga maritima 5737
-

Metals/Ions

EC Number Metals/Ions Comment 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 Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 UniProt Comment 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 Purification (Comment) 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 Comment Organism Reaction ID
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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
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
1.18.1.2 2 oxidized ferredoxin + NADPH complete reversibility of the reaction 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 Thermotoga maritima ?
-
?

Subunits

EC Number Subunits Comment Organism
1.6.1.4 heterodimer 1 * 50000 + 1 *30000, SDS-PAGE Thermotoga maritima

Synonyms

EC Number Synonyms Comment Organism
1.6.1.4 NADH-dependent reduced ferredoxin:NADP oxidoreductase
-
Thermotoga maritima
1.6.1.4 NfnAB
-
Thermotoga maritima
1.18.1.2 NADH-dependent reduced ferredoxin:NADP oxidoreductase
-
Thermotoga maritima
1.18.1.2 NfnAB
-
Thermotoga maritima

Cofactor

EC Number Cofactor Comment 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

General Information

EC Number General Information Comment 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