title: Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris.
abstract: Microsomal cytochrome P450 monooxygenases of groups 1-3 are mainly expressed in the liver and play a crucial role in phase 1 reactions of xenobiotic metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed with control of the methanol-inducible AOX1 promoter. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c by using isolated microsomes. Nearly all of the recombinant CYP was composed of the active holoenzyme, as confirmed by reduced CO difference spectra, which showed a single peak at 450 nm. Only by coexpression of human CPR and CYP was CYP2D6 activity obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion and dextrometorphan. The kinetic parameters of dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems and human microsomes. The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP activity. These recombinant strains provide a novel, easy-to-handle and cheap source for the biochemical characterisation of single microsomal cytochromes, as well as their allelic variants.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Dietrich
authors.forename: Lisa
authors.initials: L
authors.lastname: Grundmann
authors.forename: Katja
authors.initials: K
authors.lastname: Kurr
authors.forename: Laura
authors.initials: L
authors.lastname: Valinotto
authors.forename: Tanja
authors.initials: T
authors.lastname: Saussele
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Stefan
authors.initials: S
authors.lastname: Lange
mesh_terms: D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000432:Methanol / Q000378:metabolism; D020580:Methylobacterium / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase; D000432:Methanol