TY - JOUR T1 - Defining the role of the NADH-cytochrome b5 reductase 3 in the mARC-enzyme system JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.116.071845 SP - dmd.116.071845 AU - Birte Plitzko AU - Antje Havemeyer AU - Bettina Bork AU - Florian Bittner AU - Ralf Mendel AU - Bernd Clement Y1 - 2016/01/01 UR - http://dmd.aspetjournals.org/content/early/2016/07/28/dmd.116.071845.abstract N2 - 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 CYP450- and FMO-catalyzed oxidations at nitrogen centers. This enzyme system has been found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. To exert the N-reductive catalytic effect, the synergy of three components, mARC, cytochrome b5 (CYB5), and the appropriate reductase, is crucial. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein CYB5 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 siRNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in-vitro, we provide evidence that the NADH-cytochrome b5 reductase 3 (CYB5R3) is the principal reductase involved in the mARC-enzyme system and is an essential component of N-reductive metabolism in human cells. Additionally, only minimal levels of CYB5R3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase. ER -