Cloned (Comment) | Organism |
---|---|
gene 147CC514, expression of wild-type, truncated mutant, and point mutations in Escherichia coli strain C41(DE3) | Rattus norvegicus |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
C136A | site-directed mutagenesis | Rattus norvegicus |
C228A | site-directed mutagenesis | Rattus norvegicus |
C363T | site-directed mutagenesis | Rattus norvegicus |
C445L | site-directed mutagenesis | Rattus norvegicus |
C472T | site-directed mutagenesis | Rattus norvegicus |
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 |
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 |
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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics of wild-type and mutant enzymes, overview | Rattus norvegicus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
microsome | - |
Rattus norvegicus | - |
- |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
NADPH + H+ + cytochrome c | Rattus norvegicus | - |
NADP+ + reduced cytochrome c | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rattus norvegicus | P00388 | gene 147CC514 | - |
Purification (Comment) | Organism |
---|---|
recombinant truncated mutant from Escherichia coli strain C41(DE3) by aanion exchange and 2',5'-ADP-Sepharose | Rattus norvegicus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
NADPH + H+ + cytochrome c | - |
Rattus norvegicus | NADP+ + reduced cytochrome c | - |
? |
Synonyms | Comment | Organism |
---|---|---|
CYPOR | - |
Rattus norvegicus |
NADPH-cytochrome P450 oxidoreductase | - |
Rattus norvegicus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | 37 | assay at | Rattus norvegicus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | 7.7 | assay at | Rattus norvegicus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Rattus norvegicus | |
FMN | - |
Rattus norvegicus | |
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 | |
NADPH | nicotinamide binding is regulated by the Asp632 loop | Rattus norvegicus |
General Information | Comment | Organism |
---|---|---|
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 |
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 |