Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast

S. Wynne Ellis, Michael S. Ching, Philip F. Watson, Colin J. Henderson, Anthony P. Simula, Martin S. Lennard, Geoffrey T. Tucker, H. Frank Woods

    Research output: Contribution to journalComment/debate

    47 Citations (Scopus)

    Abstract

    A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50=0.05-mu-M; Qn IC,=4-mu-M) and O-demethylation (Qd IC50=0.05-mu-M; Qn IC50=4-mu-M). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.

    Original languageEnglish
    Pages (from-to)617-620
    Number of pages4
    JournalBiochemical Pharmacology
    Volume44
    Issue number4
    DOIs
    Publication statusPublished - 18 Aug 1992

    Cite this

    Ellis, S. Wynne ; Ching, Michael S. ; Watson, Philip F. ; Henderson, Colin J. ; Simula, Anthony P. ; Lennard, Martin S. ; Tucker, Geoffrey T. ; Woods, H. Frank. / Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast. In: Biochemical Pharmacology. 1992 ; Vol. 44, No. 4. pp. 617-620.
    @article{1f2439b488dc408e8889c1816c142eac,
    title = "Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast",
    abstract = "A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50=0.05-mu-M; Qn IC,=4-mu-M) and O-demethylation (Qd IC50=0.05-mu-M; Qn IC50=4-mu-M). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.",
    author = "Ellis, {S. Wynne} and Ching, {Michael S.} and Watson, {Philip F.} and Henderson, {Colin J.} and Simula, {Anthony P.} and Lennard, {Martin S.} and Tucker, {Geoffrey T.} and Woods, {H. Frank}",
    year = "1992",
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    doi = "10.1016/0006-2952(92)90394-X",
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    Ellis, SW, Ching, MS, Watson, PF, Henderson, CJ, Simula, AP, Lennard, MS, Tucker, GT & Woods, HF 1992, 'Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast', Biochemical Pharmacology, vol. 44, no. 4, pp. 617-620. https://doi.org/10.1016/0006-2952(92)90394-X

    Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast. / Ellis, S. Wynne; Ching, Michael S.; Watson, Philip F.; Henderson, Colin J.; Simula, Anthony P.; Lennard, Martin S.; Tucker, Geoffrey T.; Woods, H. Frank.

    In: Biochemical Pharmacology, Vol. 44, No. 4, 18.08.1992, p. 617-620.

    Research output: Contribution to journalComment/debate

    TY - JOUR

    T1 - Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast

    AU - Ellis, S. Wynne

    AU - Ching, Michael S.

    AU - Watson, Philip F.

    AU - Henderson, Colin J.

    AU - Simula, Anthony P.

    AU - Lennard, Martin S.

    AU - Tucker, Geoffrey T.

    AU - Woods, H. Frank

    PY - 1992/8/18

    Y1 - 1992/8/18

    N2 - A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50=0.05-mu-M; Qn IC,=4-mu-M) and O-demethylation (Qd IC50=0.05-mu-M; Qn IC50=4-mu-M). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.

    AB - A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50=0.05-mu-M; Qn IC,=4-mu-M) and O-demethylation (Qd IC50=0.05-mu-M; Qn IC50=4-mu-M). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.

    U2 - 10.1016/0006-2952(92)90394-X

    DO - 10.1016/0006-2952(92)90394-X

    M3 - Comment/debate

    VL - 44

    SP - 617

    EP - 620

    JO - Biochemical Pharmacology

    JF - Biochemical Pharmacology

    SN - 0006-2952

    IS - 4

    ER -