11 beta-hydroxylase activity in recombinant yeast mitochondria. In vivo conversion of 11-deoxycortisol to hydrocortisone.
In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.
Dumas
B
B
Biotechnology Department, ROUSSEL UCLAF, Romainville, France.
Cauet
G
G
Lacour
T
T
Degryse
E
E
Laruelle
L
L
Ledoux
C
C
Spagnoli
R
R
Achstetter
T
T
D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D000735:Androstenedione / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003345:Corticosterone / Q000378:metabolism; D003350:Cortodoxone / Q000378:metabolism*; D017931:DNA Primers; D003900:Desoxycorticosterone / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D015870:Gene Expression; D006854:Hydrocortisone / Q000378:metabolism*; D006900:Hydroxylation; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D000325:Adrenodoxin; D000735:Androstenedione; D003900:Desoxycorticosterone; D009249:NADP; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase; D003576:Electron Transport Complex IV; D003345:Corticosterone; D003350:Cortodoxone; D006854:Hydrocortisone