title: A tricistronic human adrenodoxin reductase-adrenodoxin-cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli.
abstract: Cytochrome P450 (P450) 27A1 catalyzes 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D(3), serving as an important component for the maintenance of lipid homeostasis. In eukaryotic cells P450 27A1 is a membrane-bound protein located on the inner mitochondrial membrane and requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH-adrenodoxin reductase (Adr), for catalysis in the bile acid biosynthesis pathway. A strategy was developed for the functional coexpression of P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins) in Escherichia coli, mimicking the mitochondrial P450 system. Intact bacterial cells coexpressing the P450 vector (pTC27A1) efficiently hydroxylated cholesterol at the 27 position as well as vitamin D(3) at the 25 position when supplemented with glycerol as a carbon source. Thus, E. coli containing pTC27A1 is able to hydroxylate cholesterol in a self-sufficient fashion and is suitable for further applications of protein interaction, drug discovery, and inhibitor evaluation and for the study of other mitochondrial P450s and oxysterol production in microorganisms without a need for membrane reconstitution, membrane simulation by detergents, or purification of the components.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
authors.forename: S Giovanna
authors.initials: SG
authors.lastname: Salamanca-Pinzón
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002762:Cholecalciferol / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D002762:Cholecalciferol; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D005287:Ferredoxin-NADP Reductase