The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine

Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

doi:10.1074/jbc.m115.640052

Cited by 25 publications

(29 citation statements)

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“…Oxidative radicals promote generation of hydroxylated nucleobases (69). Knockdown of mitochondrial amidoxime reducing component 2 sensitizes HeLa cells to aminopurine (70), indicating that HeLa cells constitutively express machinery to cope with hydroxylated aminopurines. IFN-␥ treatment also elevates generation of oxidative radicals (71), suggesting that this could lead to the generation of hydroxylated nucleobases in IFN-␥-treated HeLa cells.…”

Section: Discussionmentioning

confidence: 99%

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

et al. 2016

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cChlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts L-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-␥)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-␥-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells. Chlamydia trachomatis has a characteristic biphasic developmental cycle in cell culture (1). The two dominant chlamydial forms are elementary bodies (EBs) and reticulate bodies (RBs) (1). EBs represent the infectious, nonreplicative form capable of invading host cells (1). Following entry, EBs differentiate into RBs that replicate inside parasitophorous vacuoles termed inclusions (1). Maturing RBs then redifferentiate back to EBs and exit the host cell via lysis or extrusion (2). Morphologically aberrant RBs have been observed in clinical specimens (3). This abnormal developmental cycle can be recapitulated in a laboratory setting by exposing C. trachomatis to various stressors such as host cytokines (4-6), excess amino acids (7), iron deficiency (8, 9), virus coinfections (10), and antibiotics (11, 12) (reviewed in reference 13). Under these conditions, normal C. trachomatis RBs can transition into persistent forms that do not divide, are enlarged, and have aberrant morphology (13). Upon removal of the persistence-inducing stressor, RBs transition back into normal RBs and continue normal development (13). Aberrant RB morphology is a shared feature of multiple C. trachomatis persistence models and may reflect activation of a common stress response program (13). Thus, characterizing how C. trachomatis enters, survives, and reactivates from persistence could reveal new insights into chlamydial pathogenesis.The Th1 cytokine interferon gamma (IFN-␥) can induce dif...

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Section: Discussionmentioning

confidence: 99%

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

et al. 2016

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“…As the reductive counterpart to the cytochrome P450-and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers, this system plays an important role in N-reductive metabolism and has been shown to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs. Although its physiologic substrates, and therefore its physiologic functions, are largely unknown, the N-reductive system is assumed to be involved in detoxification of mutagenic and toxic aromatic hydroxylamines and regulation of the L-arginine-dependent biosynthesis of nitric oxide (Kotthaus et al, 2011;Plitzko et al, 2015). Studies have also suggested that the mARC system plays an important role in lipid synthesis (Neve et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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

Plitzko

Havemeyer

Bork

et al. 2016

Drug Metabolism and Disposition

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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.

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“…mARC therefore plays a pivotal role as a counterpart to CYP-and FMO-mediated oxygenation reactions in metabolic cycles. Furthermore, recent studies suggest that mARC is important for organisms to ensure reductive detoxification strategies, for example of toxic hydroxylamines (4) or mutagenic N-hydroxylated nucleobases (5,12). After its discovery, subsequent studies have shown that the enzyme is able to reduce the full range of Noxygenated compounds, including the capacity to reduce inorganic nitrite to nitric oxide (13) and N ω -hydroxy-L-arginine to arginine (14).…”

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confidence: 99%

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

Kubitza

Bittner

Ginsel

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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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.

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The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine

Cited by 25 publications

References 48 publications

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

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

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

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