title: Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.
abstract: The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.).
authors.forename: Birte
authors.initials: B
authors.lastname: Plitzko
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.).
authors.forename: Antje
authors.initials: A
authors.lastname: Havemeyer
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.).
authors.forename: Bettina
authors.initials: B
authors.lastname: Bork
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.).
authors.forename: Florian
authors.initials: F
authors.lastname: Bittner
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.).
authors.forename: Ralf
authors.initials: R
authors.lastname: Mendel
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts University of Kiel, Kiel, Germany (B.P., A.H., B.C.); and Department of Plant Biology, Braunschweig University of Technology, Braunschweig, Germany (B.B., F.B., R.M.) bclement@pharmazie.uni-kiel.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Clement
mesh_terms: D042966:Cytochrome-B(5) Reductase / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010091:Oximes / Q000378:metabolism*
chemical_list: D010091:Oximes; C000608634:amidoxime; D009243:NAD; D042966:Cytochrome-B(5) Reductase