Phe120 contributes to the regiospecificity of cytochrome P450 2D6

Mutation leads to the formation of a novel dextromethorphan metabolite

Jack U. Flanagan, Jean Didier Maréchal, Richard Ward, Carol A. Kemp, Lesley A. McLaughlin, Michael J. Sutcliffe, Gordon C.K. Roberts, Mark J.I. Paine, C. Roland Wolf

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

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 Km 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.

Original languageEnglish
Pages (from-to)353-360
Number of pages8
JournalBiochemical Journal
Volume380
Issue number2
DOIs
Publication statusPublished - 1 Jun 2004

Fingerprint

Dextromethorphan
Cytochrome P-450 CYP2D6
Metabolites
Mutation
Catalytic Domain
N Demethylating Oxidoreductases
NADPH-Ferrihemoprotein Reductase
Hydroxylation
Structural Models
Substrates
Cytochromes
Histidine
Leucine
Tryptophan
Alanine
Methionine
Serine
Tyrosine
Nitrogen
Ligands

Keywords

  • Cytochrome P450
  • Cytochrome P450 2D6 (CYP2D6)
  • Dextromethorphan
  • Drug metabolism
  • Ligand-binding property
  • Mutagenesis

Cite this

Flanagan, Jack U. ; Maréchal, Jean Didier ; Ward, Richard ; Kemp, Carol A. ; McLaughlin, Lesley A. ; Sutcliffe, Michael J. ; Roberts, Gordon C.K. ; Paine, Mark J.I. ; Wolf, C. Roland. / Phe120 contributes to the regiospecificity of cytochrome P450 2D6 : Mutation leads to the formation of a novel dextromethorphan metabolite. In: Biochemical Journal. 2004 ; Vol. 380, No. 2. pp. 353-360.
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abstract = "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 Km 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.",
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Flanagan, JU, Maréchal, JD, Ward, R, Kemp, CA, McLaughlin, LA, Sutcliffe, MJ, Roberts, GCK, Paine, MJI & Wolf, CR 2004, 'Phe120 contributes to the regiospecificity of cytochrome P450 2D6: Mutation leads to the formation of a novel dextromethorphan metabolite', Biochemical Journal, vol. 380, no. 2, pp. 353-360. https://doi.org/10.1042/BJ20040062

Phe120 contributes to the regiospecificity of cytochrome P450 2D6 : Mutation leads to the formation of a novel dextromethorphan metabolite. / Flanagan, Jack U.; Maréchal, Jean Didier; Ward, Richard; Kemp, Carol A.; McLaughlin, Lesley A.; Sutcliffe, Michael J.; Roberts, Gordon C.K.; Paine, Mark J.I.; Wolf, C. Roland.

In: Biochemical Journal, Vol. 380, No. 2, 01.06.2004, p. 353-360.

Research output: Contribution to journalArticle

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T1 - Phe120 contributes to the regiospecificity of cytochrome P450 2D6

T2 - Mutation leads to the formation of a novel dextromethorphan metabolite

AU - Flanagan, Jack U.

AU - Maréchal, Jean Didier

AU - Ward, Richard

AU - Kemp, Carol A.

AU - McLaughlin, Lesley A.

AU - Sutcliffe, Michael J.

AU - Roberts, Gordon C.K.

AU - Paine, Mark J.I.

AU - Wolf, C. Roland

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N2 - 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 Km 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.

AB - 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 Km 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.

KW - Cytochrome P450

KW - Cytochrome P450 2D6 (CYP2D6)

KW - Dextromethorphan

KW - Drug metabolism

KW - Ligand-binding property

KW - Mutagenesis

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