Literature summary for 1.6.2.4 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.

Cloned(Commentary)

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

Crystallization (Commentary)

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

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

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 [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

Localization	Comment	Organism	GeneOntology No.	Textmining
microsome	-	Rattus norvegicus	-	-

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Rattus norvegicus	P00388	gene 147CC514	-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

Synonyms

Synonyms	Comment	Organism
CYPOR	-	Rattus norvegicus
NADPH-cytochrome P450 oxidoreductase	-	Rattus norvegicus

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
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

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