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Literature summary extracted from

  • Sarapusit, S.; Xia, C.; Misra, I.; Rongnoparut, P.; Kim, J.J.
    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.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.6.2.4 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)

EC Number Cloned (Comment) Organism
1.6.2.4 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

EC Number Protein Variants Comment Organism
1.6.2.4 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
1.6.2.4 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
1.6.2.4 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
1.6.2.4 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

EC Number Inhibitors Comment Organism Structure
1.6.2.4 2'-AMP dead-end inhibitor Anopheles minimus
1.6.2.4 NADP+ product inhibitor Anopheles minimus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.2.4 0.0084
-
 NADH pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) Anopheles minimus
1.6.2.4 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
1.6.2.4 0.0192
-
 NADPH pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) Anopheles minimus
1.6.2.4 0.0372
-
 ferricytochrome c pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) Anopheles minimus
1.6.2.4 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
1.6.2.4 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]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.6.2.4 73000
-
 NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY), gel filtration Anopheles minimus

Organism

EC Number Organism UniProt Comment Textmining
1.6.2.4 Anopheles minimus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.6.2.4 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.2.4 2 ferricyanide + NADPH
-
 Anopheles minimus 2 ferrocyanide + NADP+ + H+
-
 ?
1.6.2.4 2 ferricytochrome c + NADH
-
 Anopheles minimus 2 ferrocytochrome c + NAD+ + H+
-
 ?
1.6.2.4 2 ferricytochrome c + NADPH
-
 Anopheles minimus 2 ferrocytochrome c + NADP+ + H+
-
 ?
1.6.2.4 ferricytochrome c + NADPH + H+
-
 Anopheles minimus ferrocytochrome c + NADP+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.6.2.4 CYPOR
-
 Anopheles minimus
1.6.2.4 NADPH-cytochrome P450 oxidoreductase
-
 Anopheles minimus

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.2.4 FAD wild-type enzyme contains 1.05 FAD Anopheles minimus
1.6.2.4 FMN wild-type enzyme contains 0.92 FMN Anopheles minimus
1.6.2.4 NADH
-
 Anopheles minimus
1.6.2.4 NADPH
-
 Anopheles minimus

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.6.2.4 0.0322
-
 NADP+ pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) Anopheles minimus
1.6.2.4 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
1.6.2.4 0.0711
-
 2'-AMP pH 7.7, 25°C, NADPH-cytochrome c oxidoreductase lacking the first 55 amino acid residues (DELTA55AnCY) Anopheles minimus
1.6.2.4 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