1521-009X/42/4/718–725$25.00 http://dx.doi.org/10.1124/dmd.113.055202 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 42:718–725, April 2014 Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics Functional Characterization of Protein Variants Encoded by Nonsynonymous Single Nucleotide Polymorphisms in MARC1 and MARC2 in Healthy Caucasians Gudrun Ott, Debora Reichmann, Cornelia Boerger, Ingolf Cascorbi, Florian Bittner, Ralf-Rainer Mendel, Thomas Kunze, Bernd Clement, and Antje Havemeyer Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany (G.O., T.K., B.C., A.H.); Department of Plant Biology, Technical University of Braunschweig, Braunschweig, Germany (D.R., F.B., R.-R.M.); and Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany (C.B., I.C.) Received September 27, 2013; accepted January 14, 2014 ABSTRACT Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b 5 type B, and NADH cytochrome b 5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds. Genetic variations are known, but their functional relevance is unclear. Our study aimed to investigate the incidence of nonsynonymous single nucleotide polymorphisms (SNPs) in the mARC genes in healthy Caucasian volunteers, to determine saturation of the protein variants with molyb- denum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype fre- quencies of six SNPs in the mARC genes (c.493A>G, c.560T>A, c.736T>A, and c.739G>C in MARC1; c.730G>A and c.735T>G in MARC2) were determined by pyrosequencing in a cohort of 340 healthy Caucasians. Protein variants were expressed in Escherichia coli. Saturation with Moco was determined by measurement of molybdenum by inductively coupled mass spectrometry. Steady state assays were performed with benzamidoxime. The six variants were of low frequency in this Caucasian population. Only one homozygous variant (c.493A; MARC1) was detected. All protein variants were able to bind Moco. Steady state assays showed statistically significant decreases of catalytic efficiency values for the mARC-2 wild type compared with the mARC-1 wild type (P < 0.05) and for two mARC-2 variants compared with the mARC-2 wild type (G244S, P < 0.05; C245W, P < 0.05). After simultaneous substitution of more than two amino acids in the mARC-1 protein, N-reductive activity was de- creased 5-fold. One homozygous variant of MARC1 was detected in our sample. The encoded protein variant (A165T) showed no different kinetic parameters in the N-reduction of benzamidoxime. Introduction Mitochondrial amidoxime reducing component (mARC) was dis- covered in 2006, and is the fourth molybdenum-containing enzyme in humans (Havemeyer et al., 2006). There are two known isoforms of mARC: mARC-1 and mARC-2. These isoforms are encoded by two genes (MARC1, NM_022746.3; and MARC2, NM_017898.3), which are located on chromosome 1 (1q41) in a tandem arrangement. Their sequences show 66% identity and 80% similarity (Wahl et al., 2010). The mARC proteins contain a mitochondrial targeting signal sequence, a predicted b-barrel domain, and a molybdenum cofactor sulfurase C-terminal domain- containing (MOSC) domain (Anantharaman and Aravind, 2002). The MOSC domain is characterized by a con- served cysteine in positions 273 and 272 of mARC-1 and mARC-2, respectively (Anantharaman and Aravind, 2002; Hille et al., 2011). mARC proteins contain molybdenum, which requires coordination by a pyranopterin to gain biologic activity. This complex is named molybdenum cofactor (Moco) and serves as a prosthetic group of mARC. Eukaryotic molybdenum enzymes are classified by the co- ordination chemistry of the molybdenum atom of Moco in two groups: the xanthine oxidoreductase family and the sulfite oxidase family. mARC proteins are regarded as members of the sulfite oxidase family (Chamizo- Ampudia et al., 2011; Havemeyer et al., 2011; Rajapakshe et al., 2011; Mendel and Kruse, 2012). The subcellular localization of mARC proteins is not fully defined (Hille et al., 2011). Previous studies in tissues of different species showed that at least one isoform of mARC was detectable in mitochondria (Da Cruz et al., 2003; Havemeyer et al., 2006; Wahl et al., 2010; Havemeyer et al., 2011; Krompholz et al., 2012; Neve et al., 2012). mARC-2 is also localized in peroxisomes (Islinger et al., 2007; Wiese et al., 2007). Studies with human cell lines revealed that mARC-1 is associated with the outer mitochondrial membrane with an N (in) -C (out) membrane orientation. The catalytic domain is localized at the C-terminus and exposed to the cytosol (Klein et al., 2012). Molybdenum-containing enzymes in general show redox activity (Hille et al., 2011). mARC-1 and mARC-2 exert N-reductive activity together with cytochrome b 5 type B (CYB5B, NM_030579.2) and NADH cytochrome b 5 reductase (CYB5R3, NM_007326.4) in the This research was supported by the Deutsche Forschungsgemeinschaft [Grants Cl-56/9-1 and ME-1266/24-1]. dx.doi.org/10.1124/dmd.113.055202. ABBREVIATIONS: ANOVA, analysis of variance; CYB5B, cytochrome b 5 type B (outer mitochondrial membrane); CYB5R3, cytochrome b 5 reductase 3; HPLC, high-performance liquid chromatography; ICP-MS, inductively coupled mass spectrometry; mARC, mitochondrial amidoxime reducing component; Moco, molybdenum cofactor; SNP, single nucleotide polymorphism; WT, wild-type. 718 at ASPET Journals on April 13, 2016 dmd.aspetjournals.org Downloaded from