RESUMEN

N-(4-Aminobutyl)-N'-(2-methoxyethyl)guanidine (8a) is a potent inhibitor targeting the hDDAH-1 active site (Ki = 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 8a-bound hDDAH-1 illuminated a unique binding mode. Together with its developed N-hydroxyguanidine prodrug 11, 8a will serve as a most widely applicable, pharmacological tool to target DDAH-1-associated diseases.

Asunto(s)

Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Guanidinas/química , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Guanidinas/síntesis química , Guanidinas/metabolismo , Humanos , Unión Proteica

RESUMEN

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 this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet-induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.

Asunto(s)

Metabolismo Energético , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Obesidad/etiología , Obesidad/genética , Oxidación-Reducción

RESUMEN

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. However, the 3D structure of this enzyme was unknown. Here we present the high-resolution crystal structure of human mARC. We give detailed insight into the coordination of its molybdenum cofactor (Moco), the catalytic mechanism, and its ability to reduce a wide range of N-oxygenated compounds. The identification of two key residues will allow future discrimination between mARC paralogs and ensure correct annotation. Since our structural findings contradict in silico predictions that are currently made by online databases, we propose domain definitions for members of the superfamily of Moco sulfurase C-terminal (MOSC) domain-containing proteins. Furthermore, we present evidence for an evolutionary role of mARC for the emergence of the xanthine oxidase protein superfamily. We anticipate the hereby presented crystal structure to be a starting point for future descriptions of MOSC proteins, which are currently poorly structurally characterized.

Asunto(s)

Proteínas Mitocondriales/química , Proteínas Mitocondriales/ultraestructura , Oxidorreductasas/química , Oxidorreductasas/ultraestructura , Catálisis , Coenzimas , Cristalografía por Rayos X/métodos , Células Eucariotas/metabolismo , Humanos , Metaloproteínas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Molibdeno/metabolismo , Cofactores de Molibdeno , Oxidación-Reducción , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Pteridinas

RESUMEN

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 N-oxygenated structures. This three component enzyme system plays a major role in N-reductive drug metabolism. Belonging to the group of N-hydroxylated structures, hydroxamic acids are also potential substrates of the mARC-system. Hydroxamic acids show a variety of pharmacological activities and are therefore often found in drug candidates. They can also exhibit toxic properties as is the case for many aryl hydroxamic acids formed during the metabolism of arylamides. Biotransformation assays using recombinant human proteins, subcellular porcine tissue fractions as well as human cell culture were performed. Here the mARC-dependent reduction of the model compound benzhydroxamic acid is reported in addition to the reduction of three drugs. In comparison with other known substrates of the molybdenum depending enzyme system (e.g., amidoxime prodrugs) the conversion rates measured here are slower, thereby reflecting the mediocre metabolic stability and oral bioavailability of distinct hydroxamic acids. Moreover, the toxic N-hydroxylated metabolite of the analgesic phenacetin, N-hydroxyphenacetin, is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs to be detoxified by other pathways. This work highlights the need to monitor the N-reductive metabolism of new drug candidates by the mARC-system when evaluating the metabolic stability of hydroxamic acid-containing structures or the potential risks of toxic metabolites.

Asunto(s)

Ácidos Hidroxámicos/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Biotransformación , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Humanos , Oxidación-Reducción , Receptor EphB3 , Porcinos

RESUMEN

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. Two of them contained either an N-terminal or a C-terminal T4L moiety fused to hmARC, while the other two contained T4L as an internal fusion partner tethered to the hmARC enzyme between two predicted secondary-structure elements. One of these internal fusion constructs could be expressed and crystallized successfully. The hmARC-T4L crystals diffracted to 1.7 Šresolution using synchrotron radiation and belonged to space group P212121 with one molecule in the asymmetric unit. Initial attempts to solve the structure by molecular replacement using T4L did not result in electron-density distributions that were sufficient for model building and interpretation of the hmARC moiety. However, this study emphasizes the utility of the T4L fusion-protein approach, which can be used for the crystallization and structure determination of membrane-bound proteins as well as soluble proteins.

Asunto(s)

Coenzimas/química , Metaloproteínas/química , Proteínas Mitocondriales/química , Muramidasa/química , Oxidorreductasas/química , Fragmentos de Péptidos/química , Pteridinas/química , Secuencia de Aminoácidos , Coenzimas/genética , Cristalización/métodos , Humanos , Metaloproteínas/genética , Proteínas Mitocondriales/genética , Cofactores de Molibdeno , Muramidasa/genética , Oxidorreductasas/genética , Fragmentos de Péptidos/genética , Difracción de Rayos X/métodos

RESUMEN

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. TMAO is an oxidation product of the hepatic flavin-containing monooxygenases (FMO), mainly of isoform 3, and it is conceivable that humans also have an enzyme reversing this toxification by reducing TMAO to its precursor trimethylamine (TMA). All prokaryotic enzymes that use TMAO as a substrate have molybdenum-containing cofactors in common. Such molybdenum-containing enzymes also exist in mammals, with the so-called mitochondrial amidoxime reducing component (mARC) representing the most recently discovered mammalian molybdenum enzyme. The enzyme has been found to exist in two isoforms, mARC1 and mARC2, both being capable of reducing a variety of N-oxygenated compounds, including nonphysiological N-oxides. To investigate whether the two isoforms of this enzyme are able to reduce and detoxify TMAO, we developed a suitable analytical method and tested TMAO reduction with a recombinant enzyme system. We found that one of the two recombinant human mARC proteins, namely, hmARC1, reduces TMAO to TMA. The N-reductive activity is relatively low and identified via the kinetic parameters with Km = (30.4 ± 9.8) mM and Vmax = (100.5 ± 12.2) nmol/(mg protein·min). Nevertheless, the ubiquitous tissue expression of hmARC1 allows a continuous reduction of TMAO whereas the counter-reaction, the production of TMAO through FMO3, can take place only in the liver where FMO3 is expressed. TMAO reduction in porcine liver subfractions showed the characteristic enrichment of N-reductive activity in the outer mitochondrial membrane. TMAO reduction was also found in human cell cultures. These findings indicate the role of hmARC1 in the metabolomic pathway of TMAO, which might contribute to the prevention of CVD. This also hints at a physiological function of the molybdenum enzyme, which remains mainly unknown to date.

Asunto(s)

Metilaminas/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Línea Celular Tumoral , Humanos , Inactivación Metabólica , Hígado/metabolismo , Metilaminas/química , Mitocondrias/metabolismo , Oxidación-Reducción , Porcinos

RESUMEN

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 synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.

Asunto(s)

Citocromo-B(5) Reductasa/metabolismo , Mitocondrias/enzimología , NAD/metabolismo , Oximas/metabolismo , Células HEK293 , Humanos

RESUMEN

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. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

Asunto(s)

Adenina/análogos & derivados , Adenosina/análogos & derivados , Proteínas de la Membrana/metabolismo , Fase I de la Desintoxicación Metabólica , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Apoptosis/genética , Citocromo-B(5) Reductasa/genética , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mitocondrias/enzimología , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo

RESUMEN

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. In the presence of NADH, mARC proteins exert N-reductive activity together with the two electron transport proteins cytochrome b5 type B and NADH cytochrome b5 reductase. This enzyme system is capable of reducing a great variety of N-hydroxylated substrates. It plays a decisive role in the activation of prodrugs containing an amidoxime structure, and in detoxification pathways, e.g., of N-hydroxylated purine and pyrimidine bases. It belongs to a group of drug metabolism enzymes, in particular as a counterpart of P450 formed N-oxygenated metabolites. Its physiological relevance, on the other hand, is largely unknown. The aim of this article is to summarize our current knowledge of these proteins with a special focus on the mammalian enzymes and their N-reductive activity.

Asunto(s)

Coenzimas/química , Proteínas de la Membrana/química , Metaloproteínas/química , Proteínas Mitocondriales/química , Molibdeno/química , Oxidorreductasas/química , Pteridinas/química , Animales , Citocromos b5/química , Citocromos b5/metabolismo , Transporte de Electrón , Humanos , Mamíferos , Proteínas de la Membrana/metabolismo , Fase I de la Desintoxicación Metabólica , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Molibdeno/metabolismo , Cofactores de Molibdeno , NAD/química , Oxidorreductasas/metabolismo

RESUMEN

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 enzyme kinetic parameters KM and Vmax for the N-reduction of ten para-substituted derivatives of the model compound benzamidoxime were determined by incubation with recombinant proteins and subcellular fractions from pig liver followed by quantification of the metabolites by HPLC. A clear influence of the substituents at position 4 on the chemical properties of the amidoxime function was confirmed by correlation analyses of (1) H NMR chemical shifts and the redox potentials of the 4-substituted benzamidoximes with Hammett's σ. However, no clear relationship between the kinetic parameters for the enzymatic reduction and Hammett's σ or the lipophilicity could be found. It is thus concluded that these properties as well as the redox potential of the amidoxime can be largely ignored during the development of new amidoxime prodrugs, at least regarding prodrug activation.

Asunto(s)

Benzamidinas/química , Oxidorreductasas/metabolismo , Profármacos/química , Amidinas/química , Amidinas/metabolismo , Animales , Benzamidinas/metabolismo , Biocatálisis , Humanos , Cinética , Hígado/metabolismo , Mitocondrias/enzimología , Molibdeno/química , Molibdeno/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Profármacos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Porcinos

RESUMEN

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 aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.

Asunto(s)

Mitocondrias/metabolismo , Sulfametoxazol/análogos & derivados , Sustitución de Aminoácidos , Animales , Biocatálisis , Cromatografía Líquida de Alta Presión , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Células HEK293 , Humanos , Cinética , Hígado/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sulfametoxazol/química , Sulfametoxazol/metabolismo , Porcinos

RESUMEN

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. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.

Asunto(s)

Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Dieta Alta en Grasa , Ayuno , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Benzamidinas/metabolismo , Línea Celular Tumoral , Activación Enzimática , Humanos , Hiperfagia/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones

RESUMEN

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. In addition, these structures open up further options for serving as prodrugs. Thus, with respect to these reactions, testing of candidates with N-oxygenated structures should not solely be carried out in microsomal enzyme sources but as well in mitochondria. However, differences in the reduction of oximes and N-oxides between the two isoforms, namely mARC1 and mARC2, were detectable; N-oxides are exclusively reduced by mARC1. We therefore assume differences between the so far unknown 3D structures of the two proteins.

Asunto(s)

Hidrazonas/química , Mitocondrias/efectos de los fármacos , Óxidos/farmacología , Oximas/farmacología , Mitocondrias/metabolismo

RESUMEN

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.

Asunto(s)

Coenzimas/metabolismo , Metaloproteínas/metabolismo , Proteínas Mitocondriales/genética , Oxidorreductasas/genética , Polimorfismo de Nucleótido Simple , Pteridinas/metabolismo , Adulto , Anciano , Benzamidinas/metabolismo , Biotransformación , Catálisis , Escherichia coli/genética , Femenino , Frecuencia de los Genes , Voluntarios Sanos , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Cofactores de Molibdeno , Unión Proteica , Población Blanca

RESUMEN

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. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

Asunto(s)

Citocromos b5/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Oximas/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Citocromo-B(5) Reductasa/genética , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Molibdeno/metabolismo , Mutación , Oxidación-Reducción , Oxidorreductasas/genética , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN

RESUMEN

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. Upamostat is the orally available amidoxime- (i.e. hydroxyamidine-) prodrug of the pharmacologically active form, WX-UK1. In this study, the reductive enzymatic activation of upamostat to its corresponding amidine WX-UK1 was analyzed. The recently discovered molybdenum enzyme "mitochondrial Amidoxime Reducing Component" (mARC) catalyses together with its electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase the reduction of N-hydroxylated prodrugs. In vitro biotransformation assays with porcine subcellular fractions and the reconstituted human enzymes demonstrate an mARC-dependent N-reduction of upamostat.

Asunto(s)

Antineoplásicos/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Fenilalanina/análogos & derivados , Piperazinas/metabolismo , Sulfonamidas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Activación Enzimática , Humanos , Oxidación-Reducción , Oximas , Fenilalanina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Porcinos

RESUMEN

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. DDAH activity (measured as L-citrulline formation from the conversion of methylarginines and alternative DDAH substrates) was detected in DDAH-1-rich porcine tissue types, that is, kidney, liver, and brain, but not in DDAH-2-rich porcine fractions, that is, spleen and thyroid. Furthermore, several ex vivo studies showed DDAH activity to be important for L-citrulline formation in porcine tissue and indicated the absence of an endogenous DDAH inhibitor in porcine tissue. This study provides new insights into tissue distributions as well as substrate selectivity for both DDAH isoforms. Although DDAH-1 is known to metabolize the endogenous NOS inhibitors L-NMMA and ADMA, a physiological function for DDAH-2 has yet to be determined. Hence, determining DDAH activity by methylarginine conversion is not suitable for analyzing isoform selectivity of DDAH-1 inhibitors as postulated.

Asunto(s)

Amidohidrolasas/metabolismo , Arginina/química , Arginina/metabolismo , Pruebas de Enzimas/métodos , Amidohidrolasas/antagonistas & inhibidores , Animales , Citrulina/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Bazo/enzimología , Porcinos , Glándula Tiroides/enzimología

RESUMEN

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. Here, we demonstrate the reductive detoxification of toxic and mutagenic N-hydroxylated nucleobases and their corresponding nucleosides by the mammalian mARC-containing enzyme system. The N-reductive activity was found in all tested tissues with the highest detectable conversion rates in liver, kidney, thyroid, and pancreas. According to the presumed localization, the N-reductive activity is most pronounced in enriched mitochondrial fractions. In vitro assays with the respective recombinant three-component enzyme system show that both mARC isoforms are able to reduce N-hydroxylated base analogues, with mARC1 representing the more efficient isoform. On the basis of the high specific activities with N-hydroxylated base analogues relative to other N-hydroxylated substrates, our data suggest that mARC proteins might be involved in protecting cellular DNA from misincorporation of toxic N-hydroxylated base analogues during replication by converting them to the correct purine or pyrimidine bases, respectively.

Asunto(s)

Adenina/análogos & derivados , Citidina/análogos & derivados , Citosina/análogos & derivados , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Adenina/química , Adenina/metabolismo , Adenina/toxicidad , Biocatálisis , Citidina/química , Citidina/metabolismo , Citidina/toxicidad , Citosina/química , Citosina/metabolismo , Citosina/toxicidad , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/aislamiento & purificación , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

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. All hitherto analyzed mammalian genomes harbor two mARC genes: molybdenum cofactor (Moco) sulferase C-terminal domain MOSC1 and MOSC2. Proteins encoded by these genes represent the simplest eukaryotic molybdenum enzymes, in that they bind only the Moco. It is also suggested that they are members of a new family of molybdenum enzymes. mARC and its N-reductive enzyme system plays a major role in drug metabolism, especially in the activation of so-called "amidoxime-prodrugs" and in the detoxification of N-hydroxylated xenobiotics, though its physiological relevance is largely unknown.

Asunto(s)

Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Biotransformación , Coenzimas/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Metaloproteínas/metabolismo , Proteínas Mitocondriales/genética , Cofactores de Molibdeno , Oxidación-Reducción , Oxidorreductasas/genética , Profármacos/metabolismo , Pteridinas/metabolismo , Especificidad por Sustrato

RESUMEN

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. It has also been shown that NOHA is reduced by microsomes and mitochondria to L-arginine. In the present study, we show for the first time that both human isoforms of the newly identified mARC (mitochondrial amidoxime reducing component) enhance the rate of reduction of NOHA, in the presence of NADH cytochrome b5 reductase and cytochrome b5, by more than 500-fold. Consequently, these results provide the first hints that mARC might be involved in mitochondrial NOHA reduction and could be of physiological significance in affecting endogenous nitric oxide levels. Possibly, this reduction represents another regulative mechanism in the complex regulation of nitric oxide biosynthesis, considering a mitochondrial NOS has been identified. Moreover, this reduction is not restricted to NOHA since the analogous arginase inhibitor NHAM (N(ω)-hydroxy-N(δ)-methyl-L-arginine) is also reduced by this system.

Asunto(s)

Arginina/análogos & derivados , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Arginina/metabolismo , Benzamidinas , Células Hep G2 , Humanos , Oxidación-Reducción , Porcinos