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.

Genome Mining in Sorangium cellulosum So ce56

“…The resulting flavin-containing supernatant was concentrated by evaporation and afterwards diluted with methanol to a final concentration of 10 M. Five l of the flavin solution were analyzed by LC-MS (Agilent 1100 series, Bruker HCT plus Ion Trap mass spectrometer). Chromatography and identification of the flavin species was performed as described previously (27).…”

Functional Characterization of Fdx1: Evidence for an Evolutionary Relationship between P450-Type and ISC-Type Ferredoxins

“…8 However, a putative ISC-type ferredoxin from the lower eukaryote Schizosaccharomyces pombe (Etp1 fd ) is known to be able to support the catalytic activity of mammalian class I cytochrome P450 enzymes. [12][13][14] Therefore, we wondered if it would be possible to identify a prokaryotic ISC-type ferredoxin able to functionally interact with a mammalian class I cytochrome P450 and whether this could provide insights into the evolutionary relationship of ISC-type and P450-type ferredoxins.…”

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

“…Recombinant Adx, Etp1 fd and Arh1 were purified as described [7,21,22]. The protein concentrations were calculated with ε 414 = 9.8 (mM cm) -1 for Etp1 fd [21] and Adx with ε 450 = 11.3 (mM cm) -1 for Arh1 [7].…”

“…The protein concentrations were calculated with ε 414 = 9.8 (mM cm) -1 for Etp1 fd [21] and Adx with ε 450 = 11.3 (mM cm) -1 for Arh1 [7]. …”

“…This assumption was supported by a comprehensive microscopic study of the ORFeome project in fission yeast [6], which determined a mitochondrial localization of both electron transfer proteins, Arh1 and Etp1 fd . Very recently, we demonstrated that the ferredoxin reductase Arh1 acts, in fact, as autologous electron donor of Etp1 fd and that both proteins together provide mammalian class I cytochromes P450 with reduction equivalents in vivo and in vitro [3,7].…”