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Literature summary extracted from

  • Xia, C.; Hamdane, D.; Shen, A.L.; Choi, V.; Kasper, C.B.; Pearl, N.M.; Zhang, H.; Im, S.C.; Waskell, L.; Kim, J.J.
    Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding (2011), J. Biol. Chem., 286, 16246-16260.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.6.2.4 gene 147CC514, expression of wild-type, truncated mutant, and point mutations in Escherichia coli strain C41(DE3) Rattus norvegicus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.6.2.4 purified mutant CYPOR with an engineered disulfide bond between the FAD and FMN domains, with or without complexed NADP+, hanging drop vapour diffusion method, mixing of 0.002 ml of 15 mg/ml protein solution with 0.002 ml of reservoir solution containing 100 mM HEPES, pH 7.2, 150 mM MgCl2, and 17% PEG 335, purified protein is treated with 2 and 20 times molar excess of FMN and NADP+, respectively, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement Rattus norvegicus

Protein Variants

EC Number Protein Variants Comment Organism
1.6.2.4 C136A site-directed mutagenesis Rattus norvegicus
1.6.2.4 C228A site-directed mutagenesis Rattus norvegicus
1.6.2.4 C363T site-directed mutagenesis Rattus norvegicus
1.6.2.4 C445L site-directed mutagenesis Rattus norvegicus
1.6.2.4 C472T site-directed mutagenesis Rattus norvegicus
1.6.2.4 C566A site-directed mutagenesis, the mutant shows full catalytic activity and a 2.5fold increased Km for NADPH compared to the wild-type enzyme Rattus norvegicus
1.6.2.4 additional information generation of a truncated -56 mutant form W677X of the rat 147CC514, with Trp677 and Ser678 truncated, the mutant exhibits decreased NADP+ binding and alterations in the conformation of the NADP+-binding site Rattus norvegicus
1.6.2.4 S457A/C630A/D675N site-directed mutagenesis, catalytically inactive mutant possessing a structure almost identical to that of the wild-type Rattus norvegicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.2.4 additional information
-
 additional information steady-state kinetics of wild-type and mutant enzymes, overview Rattus norvegicus

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.6.2.4 microsome
-
 Rattus norvegicus
-
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.6.2.4 NADPH + H+ + cytochrome c Rattus norvegicus
-
 NADP+ + reduced cytochrome c
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.6.2.4 Rattus norvegicus P00388 gene 147CC514
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.6.2.4 recombinant truncated mutant from Escherichia coli strain C41(DE3) by aanion exchange and 2',5'-ADP-Sepharose Rattus norvegicus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.2.4 NADPH + H+ + cytochrome c
-
 Rattus norvegicus NADP+ + reduced cytochrome c
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.6.2.4 CYPOR
-
 Rattus norvegicus
1.6.2.4 NADPH-cytochrome P450 oxidoreductase
-
 Rattus norvegicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.6.2.4 30 37 assay at Rattus norvegicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.6.2.4 7.4 7.7 assay at Rattus norvegicus

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.2.4 FAD
-
 Rattus norvegicus
1.6.2.4 FMN
-
 Rattus norvegicus
1.6.2.4 additional information the ribityl-nicotinamide moiety of NADP+ rotates, and the nicotinamide ring is stacked on the re-side of the flavin ring poised to transfer hydride ion to the N5 atom of FAD. the AMP-PPi portion of NADP+ binds to the enzyme in the same manner as that observed in the structures of wild-type and W677X, the nicotinamide moiety adopts a very different conformation the AMP-PPi portion of NADP+ binds to the enzyme in the same manner as that observed in the structures of wild-type and W677X, the nicotinamide moiety adopts a very different conformation Rattus norvegicus
1.6.2.4 NADPH nicotinamide binding is regulated by the Asp632 loop Rattus norvegicus

General Information

EC Number General Information Comment Organism
1.6.2.4 additional information the electron transfer pathway in CYPOR begins with the obligate two electron donor, NADPH, which transfers a hydride ion to FAD, which in turn donates electrons to the FMN cofactor. The FMN hydroquinone then transfers electrons, one at a time, to its redox partners. Comparison of the structures without and with NADP+ shows movement of the Gly631-Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631-Asn635 loop to a position that permits NADP(H) binding. Comparison of mutant and wild-type structures, overview. The Gly631-Asn635 loop movement controls NADPH binding and NADP+ release, this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners Rattus norvegicus
1.6.2.4 physiological function CYPOR is an essential electron donor to microsomal P450s, therefore it is critical to the function of the large number of physiologic processes regulated by P450 Rattus norvegicus