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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1amo
Original Entry
Title:
Oxidoreductase
Compound:
Nadph-cytochrome p450 reductase
Mutant:
No
UniProt/Swiss-Prot:
P00388-NCPR_RAT
EC Class:
1.6.2.4
Other CSA Entries:
Overview of all sites for 1amo
Homologues of 1amo
Entries for UniProt/Swiss-Prot: P00388
Entries for EC: 1.6.2.4
Other Databases:
PDB entry: 1amo
PDBsum entry: 1amo
UniProt/Swiss-Prot: P00388
IntEnz entry: 1.6.2.4
Literature Report:
Introduction:
Rat NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyses the transfer of electrons from NADPH to a range of cytochrome P450 proteins. Other electron acceptors include heme oxygenase, cytochrome b5 and fatty-acid elongase. It is a 78kDa protein and is attached to the cytoplasmic side of the endoplasmic reticulum and the outer membrane of the nuclear envelope by a hydrophobic N-terminal membrane anchor.

The FAD and FMN containing domains are linked by a flexible peptide hinge and it is thought that CYPOR evolved after a fusion of genes encoding for a FMN-containing flavodoxin and a FAD- binding ferredoxin-NADP reductase.

Mechanism:
The overall reaction is the oxidation of NADPH to NADP and the reduction of cytochrome P450, or other protein substrate. Both two-electron and four-electron reduced enzyme species have been detected in vitro. It is not yet known if CYPOR is reduced to the two-, three- or four-electron form during catalytic turnover in vivo. However, it has been proposed that the electron transfer leading to the four-electron reduced species is unlikely to have any biological significance.

A hydride ion is transferrred from NADPH to FAD. FAD then transfers electrons to FMN which in turn passes them on to the heme of cytochrome P450. Electrons are transferred from FADH2 one at a time resulting in the formation of FMNH* and FADH* radical intermediates.

Sites:

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Found by:
Literature reference 
PsiBLAST alignment on 1fnb

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
SERA 457 457Sidechain
ElectrostaticCofactor
Ser457 stabilises the semiquinone form of FAD. Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11371558 Current protein Kinetic studies
PubMed ID 11371558 Current protein Conservation of residue
PubMed ID 11371558 Current protein Mutagenesis of residue
PubMed ID 11371558 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 630 630Sidechain
ElectrostaticTransition state
Cys630 stabilises the transition state carbocation of the nicotinamide ring during the hydride transfer step. Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11371558 Current protein Mutagenesis of residue
PubMed ID 11371558 Current protein Kinetic studies
PubMed ID 11371558 Current protein Conservation of residue
PubMed ID 11371558 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 675 675Sidechain
ElectrostaticResidue
ElectrostaticSubstrate
Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD. A carboxylate oxygen on Asp675 acts as the hydrogen bond acceptor for hydrogen bonding to Ser457 and Cys630 in the abasence of NADPH. When NADPH is bound Asp675 forms hydrogen bonds to its amide group instead.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11371558 Current protein Kinetic studies
PubMed ID 11371558 Current protein Conservation of residue
PubMed ID 11371558 Current protein Mutagenesis of residue
PubMed ID 11371558 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
FADA 750 0
Electron donor/acceptorCofactor
When the FAD is reduced it accepts a hydride ion from and NADPH and a proton from water. When it is oxidised it donates two protons to water and transfers two electrons to FMN. The electrons are transferred to FMN one at a time.
Evidence from paper Evidence concerns Evidence type
PubMed ID 18681889 Related protein: UniProt P16435
PubMed ID 11371558 Current protein Conservation of residue
PubMed ID 18681889 Related protein: UniProt P16435 pH dependence of reaction
PubMed ID 11371558 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 18681889 Related protein: UniProt P16435 Kinetic studies
PubMed ID 18681889 Related protein: UniProt P16435 Ligand is essential for catalysis

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
FMNA 751 0
Electron donor/acceptorSubstrate
FMN is reduced by FAD and transfers the electrons successively to the heme of the substrate. The heme is a one-electron acceptor.
Evidence from paper Evidence concerns Evidence type
PubMed ID 18681889 Related protein: UniProt P16435 pH dependence of reaction
PubMed ID 18681889 Related protein: UniProt P16435
PubMed ID 11371558 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 18681889 Related protein: UniProt P16435 Ligand is essential for catalysis
PubMed ID 11371558 Current protein Conservation of residue
PubMed ID 18681889 Related protein: UniProt P16435 Kinetic studies
Notes:


References:
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