Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes | Anabaena sp. |
Crystallization (Comment) | Organism |
---|---|
purified recombinant mutant E139K, hanging drop vapour diffusion method, 0.002 ml protein solution containing 0.75 mM protein in 10 mM Tris-HCl, pH 8.0, plus 0.001 ml reservoir solution containing 5% w/v beta-octylglycoside, 18% PEG 6000, 20 mM ammonium sulfate, and 0.1 M MES/NaOH, pH 5.5, equilibration against 1 ml reservoir solution at 20°C, phase separation due to detergent, X-ray diffraction structure determination and analysis at 2.5 A resolution | Anabaena sp. |
Protein Variants | Comment | Organism |
---|---|---|
E139D | site-directed mutagenesis, altered conformation compared to the wild-type enzyme, slightly reduced activity compared to the wild-type enzyme | Anabaena sp. |
E139K | site-directed mutagenesis, mutant enzyme shows increased interaction with ferredoxin and reduces the appropriate orientation of flavodoxin, altered conformation compared to the wild-type enzyme, increased activity compared to the wild-type enzyme | Anabaena sp. |
E139Q | site-directed mutagenesis, mutant enzyme shows increased interaction with ferredoxin and reduces the appropriate orientation of flavodoxin, altered conformation compared to the wild-type enzyme, increased activity compared to the wild-type enzyme | Anabaena sp. |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
additional information | activity decreases with elevated ionic strength | Anabaena sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | stopped-flow kinetics measurements, pH 8.0, 13°C, steady-state kinetics for wild-type and mutant enzymes dependent on ionic strength, overview | Anabaena sp. | |
0.00027 | - |
oxidized ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.0005 | - |
oxidized ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
0.0009 | - |
oxidized ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
0.0025 | - |
oxidized ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
0.0034 | - |
NADPH | recombinant mutant E139Q, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.0035 | - |
oxidized ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
0.0043 | - |
oxidized ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.0047 | - |
NADPH | recombinant mutant E139D, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.0058 | - |
NADPH | recombinant mutant E139K, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.006 | - |
NADPH | recombinant wild-type enzyme, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.02 | - |
oxidized ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
0.021 | - |
oxidized ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
0.023 | - |
oxidized ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
0.023 | - |
oxidized ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.1 | - |
oxidized ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
0.4 | - |
oxidized ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
reduced ferredoxin + NADP+ | Anabaena sp. | - |
oxidized ferredoxin + NADPH | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Anabaena sp. | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH | Glu139 is essential for interaction with the substrate steering ferredoxin, flavodoxin and NADP+/NADPH in appropriate position for docking, Glu139 is not involved in binding | Anabaena sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | NADPH-dependent cytochrome c reductase assay for determination of activity with ferredoxin or flavodoxin as electron carrier | Anabaena sp. | ? | - |
? | |
oxidized 2,6-dichlorophenolindophenol + NADP+ | diaphorase activity | Anabaena sp. | reduced 2,6-dichlorophenolindophenol + NADPH | - |
? | |
oxidized ferredoxin + NADPH + H+ | - |
Anabaena sp. | reduced ferredoxin + NADP+ | - |
r | |
reduced ferredoxin + NADP+ | - |
Anabaena sp. | oxidized ferredoxin + NADPH | - |
r | |
reduced ferredoxin + NADP+ | hydride transfer of the N5 of the FAD isoalloxazine ring to the NADP+ nicotinamide ring, transfer of 2 electrons via the one-electron-carrier ferredoxin | Anabaena sp. | oxidized ferredoxin + NADPH | - |
r | |
reduced ferredoxin + NADP+ | - |
Anabaena sp. | oxidized ferredoxin + NADPH + H+ | - |
r | |
reduced flavodoxin + NADP+ | - |
Anabaena sp. | oxidized flavodoxin + NADPH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
More | three-dimensional structure of mutant E139K | Anabaena sp. |
Synonyms | Comment | Organism |
---|---|---|
ferredoxin-NADP+ reductase | - |
Anabaena sp. |
FNR | - |
Anabaena sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Anabaena sp. |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | 8 | reduced flavodoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
14 | - |
reduced flavodoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
17 | - |
reduced flavodoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
19 | - |
reduced flavodoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
20 | - |
reduced flavodoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
24 | - |
reduced flavodoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
25 | - |
reduced flavodoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
25 | - |
reduced flavodoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
26 | - |
reduced flavodoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
38 | - |
reduced flavodoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
58 | - |
reduced ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
59 | - |
NADPH | recombinant mutant E139K, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
70 | - |
reduced ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
81 | - |
NADPH | recombinant wild-type enzyme, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
88 | - |
NADPH | recombinant mutant E139Q, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
89 | - |
NADPH | recombinant mutant E139D, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
117 | - |
reduced ferredoxin | recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
120 | - |
reduced ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
135 | - |
reduced ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
148 | - |
reduced ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 200 mM | Anabaena sp. | |
155 | - |
reduced ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
176 | - |
reduced ferredoxin | recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
192 | - |
reduced ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
209 | - |
reduced ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM | Anabaena sp. | |
225 | - |
reduced ferredoxin | recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. | |
280 | - |
reduced ferredoxin | recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM | Anabaena sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Anabaena sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | flavoenzyme | Anabaena sp. |