knockout studies with mARC 1 The abbreviations used are: ALT, alanine aminotransferase; BA, benzamidine; BAO, benzamidoxime; CYB5B, cytochrome b5 B; CYB5R, NADH-cytochrome b5 reductase; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; GGT; gamma-glutamyltransferase; HDL, high-density lipoproteins; HFD, high fat diet; i.v., intravenous; IMPC, International Mouse Phenotyping Consortium; KO, knockout; mARC, mitochondrial amidoxime reducing component; Moco, molybdenum cofactor; ND, normal diet; POI, protein of interest; TBS, tris-buffered saline; TBST, TBS containing Tween 20; TG, tryglycerides. # JBC/2019/007606R1 Mitochondrial amidoxime-reducing component 2 (mARC2) has a significant role in N-reductive activity and energy metabolism Sophia Rixen 1 , Antje Havemeyer 1 , Anita Tyl-Bielicka 2 , Kazimiera Pysniak 2 , Marta Gajewska 2 , Maria Kulecka 3 , Jerzy Ostrowski 2,3 , Michal Mikula 2 *, Bernd Clement 1 * From the 1 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Germany; 2 Department of Genetics, Maria Sklodowska-Curie Institute - Cancer Center, Warsaw, Poland; 3 Department of Gastroenterology, Hepatology and Clinical Oncology, Centre of Postgraduate Medical Education, Warsaw, Poland Running title: knockout studies with mARC Present address: Pharmaceutical Institute of the Christian-Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany *To whom correspondence should be addressed: Bernd Clement, Christian-Albrechts-University, Pharmaceutical Institute, Gutenbergstraße 76, 24118 Kiel, Germany, tel.: 049 431-8801126, fax: 049 431 8801352, email: bclement@pharmazie.uni-kiel.de Michal Mikula, Maria Sklodowska-Curie Institute - Oncology Center, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland. tel. 048 225462655, fax: 048 225462449, email: mikula.michal@coi.pl Keywords: mitochondrial amidoxime-reducing component (mARC), gene knockout, transgenic mice, drug metabolism, lipid metabolism, N-reduction, enzyme catalysis, molybdenum, energy metabolism, xenobiotic metabolism ABSTRACT The mitochondrial amidoxime-reducing component (mARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two mARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In the present study, we characterized an existing knockout (KO) mouse model lacking the functional MARC2 gene and fed a high-fat diet (HFD) and also performed in vivo and in vitro experiments to characterize reductase activity toward known mARC substrates. The MARC2-KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical mARC substrates, only a small residual reductive activity was still detectable in the MARC2-KO mice. Residual detected reductase activity in the MARC2-KO mice could be explained by mARC1 expression that was hardly unaffected by the KO, and we found no evidence for significant activities of other reductase enzymes. These results clearly indicate that mARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of the MARC2-KO mice were a lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.007606 The latest version is at JBC Papers in Press. Published on September 25, 2019 as Manuscript RA119.007606 by guest on May 28, 2020 http://www.jbc.org/ Downloaded from