An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.
The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.
Gilep
A A
AA
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus.
Guryev
O L
OL
Usanov
S A
SA
Estabrook
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013053:Spectrophotometry
D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D009251:NADPH-Ferrihemoprotein Reductase