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Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1

Item Type:Article
Title:Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1
Creators Name:Bureik, M. and Schiffler, B. and Hiraoka, Y. and Vogel, F. and Bernhardt, R.
Abstract: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).
Keywords:Adrenodoxin, Aldosterone Synthase, Bacterial Proteins, Carrier Proteins, Molecular Cloning, Cortodoxone, Cytochrome P-450 Enzyme System, Desoxycorticosterone, Electron Transport, Enzyme Activation, Enzymologic Gene Expression Regulation, Fungal Proteins, Gene Expression Fungal Regulation, Hydroxylation, Membrane Proteins, Mitochondria, Protein Sequence Analysis, Recombinant Proteins, Schizosaccharomyces, Schizosaccharomyces Pombe Proteins
Publisher:American Chemical Society
Page Range:2311-2321
Date:1 January 2002
Official Publication:https://doi.org/10.1021/bi0157870
PubMed:View item in PubMed

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