Discovery of -(4-Aminobutyl)-'-(2-methoxyethyl)guanidine as the First Selective, Nonamino Acid, Catalytic Site Inhibitor of Human Dimethylarginine Dimethylaminohydrolase-1 (DDAH-1).

-(4-Aminobutyl)-'-(2-methoxyethyl)guanidine () is a potent inhibitor targeting the DDAH-1 active site ( = 18 μM) and derived from a series of guanidine- and amidine-based inhibitors. Its nonamino acid nature leads to high selectivities toward other enzymes of the nitric oxide-modulating system. Crystallographic data of -bound DDAH-1 illuminated a unique binding mode.

Mitochondrial amidoxime-reducing component 2 (MARC2) has a significant role in -reductive activity and energy metabolism.

The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, and , which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of hydroxylated compounds.

Crystal structure of human mARC1 reveals its exceptional position among eukaryotic molybdenum enzymes.

Biotransformation enzymes ensure a viable homeostasis by regulating reversible cycles of oxidative and reductive reactions. The metabolism of nitrogen-containing compounds is of high pharmaceutical and toxicological relevance because N-oxygenated metabolites derived from reactions mediated by cytochrome P450 enzymes or flavin-dependent monooxygenases are in some cases highly toxic or mutagenic. The molybdenum-dependent mitochondrial amidoxime-reducing component (mARC) was found to be an extremely efficient counterpart, which is able to reduce the full range of N-oxygenated compounds and thereby mediates detoxification reactions.

The Involvement of the Mitochondrial Amidoxime Reducing Component (mARC) in the Reductive Metabolism of Hydroxamic Acids.

The mitochondrial amidoxime reducing component is a recently discovered molybdenum enzyme in mammals which, in concert with the electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase, catalyzes the reduction of -oxygenated structures. This three component enzyme system plays a major role in -reductive drug metabolism. Belonging to the group of -hydroxylated structures, hydroxamic acids are also potential substrates of the mARC-system.

T4 lysozyme-facilitated crystallization of the human molybdenum cofactor-dependent enzyme mARC.

The human mitochondrial amidoxime reducing component (hmARC) is a molybdenum cofactor-dependent enzyme that is involved in the reduction of a diverse range of N-hydroxylated compounds of either physiological or xenobiotic origin. In this study, the use of a fusion-protein approach with T4 lysozyme (T4L) to determine the structure of this hitherto noncrystallizable enzyme by X-ray crystallography is described. A set of four different hmARC-T4L fusion proteins were designed.

Detoxification of Trimethylamine N-Oxide by the Mitochondrial Amidoxime Reducing Component mARC.

Although known for years, the toxic effects of trimethylamine N-oxide (TMAO), a physiological metabolite, were just recently discovered and are currently under investigation. It is known that elevated TMAO plasma levels correlate with an elevated risk for cardiovascular disease (CVD). Even though there is a general consensus about the existence of a causal relationship between TMAO and CVD, the underlying mechanisms are not fully understood.

Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

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 cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism.

The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools.

Electrochemical and mARC-catalyzed enzymatic reduction of para-substituted benzamidoximes: consequences for the prodrug concept "amidoximes instead of amidines".

The mitochondrial amidoxime reducing component (mARC) activates amidoxime prodrugs by reduction to the corresponding amidine drugs. This study analyzes relationships between the chemical structure of the prodrug and its metabolic activation and compares its enzyme-mediated vs. electrochemical reduction.

The mammalian molybdenum enzymes of mARC.

The "mitochondrial amidoxime reducing component" (mARC) is the most recently discovered molybdenum-containing enzyme in mammals. All mammalian genomes studied to date contain two mARC genes: MARC1 and MARC2. The proteins encoded by these genes are mARC-1 and mARC-2 and represent the simplest form of eukaryotic molybdenum enzymes, only binding the molybdenum cofactor.

Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC).

Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated.

The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice.

The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs.

The mitochondrial amidoxime reducing component (mARC): involvement in metabolic reduction of N-oxides, oximes and N-hydroxyamidinohydrazones.

The mitochondrial amidoxime reducing component (mARC) is a molybdenum-containing enzyme and capable of reducing N-hydroxylated structures such as amidoxime prodrugs. In this study, we tested the involvement of mARC in the reduction of N-oxides (amitriptyline-N-oxide, nicotinamide-N-oxide), oximes ((E)-/(Z)-2,4,6-trimethylacetophenonoxime) and a N-hydroxyamidinohydrazone (guanoxabenz). All groups are reduced by mARC proteins, and the enzymes are therefore involved in the interconversion of N-oxygenated metabolites originating from cytochrome P450s and flavin-containing monooxygenases.

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

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.

The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells.

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs.

Activation of the anti-cancer agent upamostat by the mARC enzyme system.

Upamostat (Mesupron®) is a new small molecule serine protease inhibitor. The drug candidate was developed to inhibit the urokinase-type plasminogen activator (uPA) system, which plays a major role in tumor invasion and metastasis. Upamostat is currently in clinical development as an anti-metastatic and non-cytotoxic agent against pancreatic and breast cancer.

Dimethylarginine-dimethylaminohydrolase-2 (DDAH-2) does not metabolize methylarginines.

Free endogenous methylarginines, N(ω)-monomethyl-L-arginine (L-NMMA) and N(ω),N(ω')-dimethyl-L-arginine (ADMA), inhibit NO synthases (NOSs) and are metabolized by dimethylargininedimethylaminohydrolase (DDAH). A postulated metabolism has been shown several times for DDAH-1, but the involvement of DDAH-2 in the degradation of ADMA and L-NMMA is still a matter of debate. Determination of the isoform-specific DDAH protein expression profiles in various porcine tissue types shows a correlation of DDAH activity only with DDAH-1 levels.

The mitochondrial Amidoxime Reducing Component (mARC) is involved in detoxification of N-hydroxylated base analogues.

The "mitochondrial Amidoxime Reducing Component" (mARC) is the newly discovered fourth molybdenum enzyme in mammals. All hitherto analyzed mammals express two mARC proteins, referred to as mARC1 and mARC2. Together with their electron transport proteins cytochrome b(5) and NADH cytochrome b(5) reductase, they form a three-component enzyme system and catalyze the reduction of N-hydroxylated prodrugs.

The fourth mammalian molybdenum enzyme mARC: current state of research.

The mitochondrial amidoxime-reducing component (mARC) is a recently discovered molybdenum-containing enzyme in mammalians. Upon reconstitution with the electron transport proteins, cytochrome b(5) and its reductase, this molybdenum enzyme is capable of reducing N-hydroxylated compounds. It was named mARC because the N-reduction of amidoxime structures was initially studied using this isolated mitochondrial enzyme.

Reduction of N(ω)-hydroxy-L-arginine by the mitochondrial amidoxime reducing component (mARC).

NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (N(ω)-hydroxy-L-arginine). This intermediate is rapidly converted further, but to a small extent can also be liberated from the active site of NOSs and act as a transportable precursor of nitric oxide or potent physiological inhibitor of arginases. Thus its formation is of enormous importance for the nitric-oxide-generating system.

Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes.

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b(5), and NADH/FAD-dependent cytochrome b(5) reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1.

Identification of the missing component in the mitochondrial benzamidoxime prodrug-converting system as a novel molybdenum enzyme.

Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction.

Hepatic, extrahepatic, microsomal, and mitochondrial activation of the N-hydroxylated prodrugs benzamidoxime, guanoxabenz, and Ro 48-3656 ([[1-[(2s)-2-[[4-[(hydroxyamino)iminomethyl]benzoyl]amino]-1-oxopropyl]-4-piperidinyl]oxy]-acetic acid).

In previous studies, it was shown that liver microsomes from rabbit, rat, pig, and human are involved in the reduction of N-hydroxylated amidines, guanidines, and amidinohydrazones of various drugs and model compounds (Drug Metab Rev 34: 565-579). One responsible enzyme system, the microsomal benzamidoxime reductase, consisting of cytochrome b5, its reductase, and a cytochrome P450 isoenzyme, was isolated from pig liver microsomes (J Biol Chem 272:19615-19620). Further investigations followed to establish whether such enzyme systems are also present in microsomes of other organs such as brain, lung, and intestine.

Reduction of sulfamethoxazole and dapsone hydroxylamines by a microsomal enzyme system purified from pig liver and pig and human liver microsomes.

Biotransformation involving nitrogen are of pharmacological and toxicological relevance. In principle, nitrogen containing functional groups can undergo all the known biotransformation processes such as oxidation, reduction, hydrolysis and formation of conjugates. For the N-reduction of benzamidoxime an oxygen-insensitive liver microsomal enzyme system that required cytochrome b5, NADH-cytochrome b5 reductase and a cytochrome P450 isoenzyme of the subfamily 2D has been described.