Phe120 contributes to the regiospecificity of cytochrome P450 2D6: mutation leads to the formation of a novel dextromethorphan metabolite.
Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1' hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on K(m) values are observed for dextromethorphan O-demethylation than for bufuralol 1' hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120-->Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.
Flanagan
Jack U
JU
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
Maréchal
Jean-Didier
JD
Ward
Richard
R
Kemp
Carol A
CA
McLaughlin
Lesley A
LA
Sutcliffe
Michael J
MJ
Roberts
Gordon C K
GC
Paine
Mark J I
MJ
Wolf
C Roland
CR
D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003915:Dextromethorphan / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010649:Phenylalanine / Q000502:physiology*; D013379:Substrate Specificity / Q000235:genetics / Q000502:physiology
D007527:Isoenzymes; D010649:Phenylalanine; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; C043872:bufuralol 1'-hydroxylase