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Drug metabolism and disposition: the biological fate of chemicals

Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians.


PMID 24423752

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.