Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1.
Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations. In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450(aldo)) in fission yeast Schizosaccharomyces pombe. Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and electron microscopy demonstrated that the mitochondrial localization signal of the human protein is functional in S. pombe. The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an electron transport chain that consists of two proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells. Searching the fission yeast genome for adrenodoxin homologues, a gene was identified that codes for a protein with an amino terminal domain homologous to COX15 of Saccharomyces cerevisiae and a carboxy terminal ferredoxin domain. It was found that overexpression of this gene significantly enhances steroid hydroxylase activity of CYP11B2 expressing fission yeast cells. Moreover, the bacterially expressed ferredoxin domain of this protein can replace adrenodoxin in a reconstituted steroid hydroxylation assay and transfer electrons from adrenodoxin reductase to a mammalian or a bacterial cytochrome P450. Therefore, we suggest to name this protein etp1 (electron-transfer protein 1).
Bureik
Matthias
M
Universität des Saarlandes, FR 8.8 Biochemie, Postfach 151150, D-66041 Saarbrücken, Germany.
Schiffler
Burkhard
B
Hiraoka
Yasushi
Y
Vogel
Frank
F
Bernhardt
Rita
R
D000325:Adrenodoxin / Q000737:chemistry; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003350:Cortodoxone / Q000378:metabolism; D019405:Cytochrome P-450 CYP11B2 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003900:Desoxycorticosterone / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D006900:Hydroxylation; D008565:Membrane Proteins; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C450201:etp1 protein, S pombe; D000325:Adrenodoxin; D003900:Desoxycorticosterone; D003577:Cytochrome P-450 Enzyme System; D019405:Cytochrome P-450 CYP11B2; D003350:Cortodoxone