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Literature summary for 1.6.2.4 extracted from
- NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability (2008), Arch. Biochem. Biophys., 477, 53-59.
Application
| Application | Comment | Organism |
|---|---|---|
| medicine | triple mutant L86F/L219F/P456A or a double mutant L86F/L219F enzyme will serve as a good model of mosquito CYPOR in future structural studies and in the development of new antimalarial agents | Anopheles minimus |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) is expressed in Escherichia coli loses FMN, leading to an unstable protein and subsequent aggregation. The triple mutant L86F/L219F/P456FDELTA55AnCY exhibits increased stability with significant reduction in aggregation compared to the wild-type enzyme | Anopheles minimus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| L219F | mutant enzyme contains 0.83 FMN and 0.5 FAD compared to 0.53 FMN and 1.05 FAD for wild-type enzyme | Anopheles minimus |
| L86F | mutant enzyme contains 0.83 FMN and 0.5 FAD compared to 0.92 FMN and 1.05 FAD for wild-type enzyme | Anopheles minimus |
| L86F/L219F/P456A | increased stability with significant reduction in aggregation compared to the wild-type enzyme. The triple mutant is purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD (compared to 0.92 FMN and 1.05 FAD for wild-type enzyme). Deficiency in FAD content is overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site ping-pong mechanism with similar kinetic constants | Anopheles minimus |
| P456A | mutant enzyme contains 0.28 FMN and 0.5 FAD compared to 0.5 FMN and 1.05 FAD for wild-type enzyme | Anopheles minimus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| 2'-AMP | dead-end inhibitor | Anopheles minimus |  |
| NADP+ | product inhibitor | Anopheles minimus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0084 | - |
NADH | pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0108 | - |
NADH | pH 7.7, 25°C, triple mutant L86F/L219F/P456A of NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0192 | - |
NADPH | pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0372 | - |
ferricytochrome c | pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0425 | - |
NADPH | pH 7.7, 25°C, triple mutant L86F/L219F/P456A of NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0924 | - |
ferricytochrome c | pH 7.7, 25°C, triple mutant L86F/L219F/P456A of NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 73000 | - |
NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY), gel filtration | Anopheles minimus |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Anopheles minimus | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) and recombinant mutant enzyme L86F/L219F/P456ADELTA55AnCY | Anopheles minimus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 ferricyanide + NADPH | - |
Anopheles minimus | 2 ferrocyanide + NADP+ + H+ | - |
? | |
| 2 ferricytochrome c + NADH | - |
Anopheles minimus | 2 ferrocytochrome c + NAD+ + H+ | - |
? | |
| 2 ferricytochrome c + NADPH | - |
Anopheles minimus | 2 ferrocytochrome c + NADP+ + H+ | - |
? | |
| ferricytochrome c + NADPH + H+ | - |
Anopheles minimus | ferrocytochrome c + NADP+ | - |
r | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CYPOR | - |
Anopheles minimus |
| NADPH-cytochrome P450 oxidoreductase | - |
Anopheles minimus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | wild-type enzyme contains 1.05 FAD | Anopheles minimus | |
| FMN | wild-type enzyme contains 0.92 FMN | Anopheles minimus | |
| NADH | - |
Anopheles minimus | |
| NADPH | - |
Anopheles minimus | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0322 | - |
NADP+ | pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0459 | - |
NADP+ | pH 7.7, 25°C, triple mutant L86F/L219F/P456A of NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.0711 | - |
2'-AMP | pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
| 0.1319 | - |
2'-AMP | pH 7.7, 25°C, triple mutant L86F/L219F/P456A of NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) | Anopheles minimus | |
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