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 |
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 |
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 |
EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|---|
1.18.1.2 | cytoplasm | - |
Thermotoga maritima | 5737 | - |
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 |
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 |
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 | - |
- |
- |
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 |
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 |
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 | ? | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
1.6.1.4 | heterodimer | 1 * 50000 + 1 *30000, SDS-PAGE | Thermotoga maritima |
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 |
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 |
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 |