title: The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.
abstract: Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.
authors.forename: Kerstin M
authors.initials: KM
authors.lastname: Ewen
authors.forename: Burkhard
authors.initials: B
authors.lastname: Schiffler
authors.forename: Heike
authors.initials: H
authors.lastname: Uhlmann-Schiffler
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
mesh_terms: D000595:Amino Acid Sequence; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000502:physiology*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000502:physiology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000502:physiology*
chemical_list: D005420:Flavoproteins; D029702:Schizosaccharomyces pombe Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase