A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding.
The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.
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Department of Biochemistry, University of Leicester, United Kingdom.
Paine
M J
MJ
Sutcliffe
M J
MJ
Lian
L Y
LY
Primrose
W U
WU
Wolf
C R
CR
Roberts
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D018411:Spodoptera; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6