Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast. Implication in in vitro cytochrome p45011beta monooxygenase system.
The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.
Lacour
T
T
Biotechnology Department, Hoechst Marion Roussel, 102 route de Noisy, 93230 Romainville, France.
Achstetter
T
T
Dumas
B
B
D000325:Adrenodoxin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000378:metabolism; D004926:Escherichia coli; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008322:Mammals; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013172:Spores, Fungal; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013367:Submitochondrial Particles / Q000201:enzymology; D013379:Substrate Specificity
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D009243:NAD; D000325:Adrenodoxin; D009249:NADP; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase