title: Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians.
abstract: Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b5 type B, and NADH cytochrome b5 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 molybdenum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype frequencies 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 decreased 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.
authors.affiliation: 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.).
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ott
authors.forename: Debora
authors.initials: D
authors.lastname: Reichmann
authors.forename: Cornelia
authors.initials: C
authors.lastname: Boerger
authors.forename: Ingolf
authors.initials: I
authors.lastname: Cascorbi
authors.forename: Florian
authors.initials: F
authors.lastname: Bittner
authors.forename: Ralf-Rainer
authors.initials: RR
authors.lastname: Mendel
authors.forename: Thomas
authors.initials: T
authors.lastname: Kunze
authors.forename: Bernd
authors.initials: B
authors.lastname: Clement
authors.forename: Antje
authors.initials: A
authors.lastname: Havemeyer
mesh_terms: D000328:Adult; D000368:Aged; D001550:Benzamidines / Q000378:metabolism; D001711:Biotransformation; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005787:Gene Frequency; D064368:Healthy Volunteers; D006720:Homozygote; D006801:Humans; D008297:Male; D008667:Metalloproteins / Q000378:metabolism*; D008875:Middle Aged; D024101:Mitochondrial Proteins / Q000235:genetics*; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D011621:Pteridines / Q000378:metabolism*; D044465:White People
chemical_list: D001550:Benzamidines; D003067:Coenzymes; D008667:Metalloproteins; D024101:Mitochondrial Proteins; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases; D011621:Pteridines; C053561:benzamidoxime; C022261:molybdopterin; C554553:MTARC1 protein, human; C554552:mitochondrial amidoxime reducing component 2, human