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Sample records for ribonucleotide reductase mediate

  1. AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

    PubMed

    Kolwicz, Stephen C; Odom, Guy L; Nowakowski, Sarah G; Moussavi-Harami, Farid; Chen, Xiaolan; Reinecke, Hans; Hauschka, Stephen D; Murry, Charles E; Mahairas, Gregory G; Regnier, Michael

    2016-02-01

    Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure. PMID:26388461

  2. Ribonucleotide reductase metallocofactor: assembly, maintenance and inhibition

    PubMed Central

    ZHANG, Caiguo; LIU, Guoqi; HUANG, Mingxia

    2014-01-01

    Ribonucleotide reductase (RNR) supplies cellular deoxyribonucleotide triphosphates (dNTP) pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises α and β subunits: α contains the catalytic and allosteric sites; β houses a diferric-tyrosyl radical cofactor (FeIII2-Y•) that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high-fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryotic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metallocofactor biosynthesis and its implication in RNR-targeting therapeutics. PMID:24899886

  3. Structural features of the ribonucleotide reductase of Aujeszky's disease virus.

    PubMed

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-01-01

    A gene construct of the Aujeszky's disease virus (ADV) genome was prepared and the DNA fragment encoding the ribonucleotide reductase was structurally characterized. We determined the entire DNA sequence of two adjacent open reading frames of the ribonucleotide reductase genes with the intergenic sequence of nine base pairs. From the sequence analysis we predict that Aujeszky's disease virus encodes a ribonucleotide reductase which comprises two polypeptides--large and small subunits, with sizes of 835 and 303 amino acids, respectively. Nucleotide and amino acid sequences of the large and small subunits of the Aujeszky's disease virus ribonucleotide reductase have been compared with that of other herpesviruses, and structural features of both proteins have been characterized. PMID:7810419

  4. DNA damage induction of ribonucleotide reductase.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. Images PMID:2513480

  5. The effect of copper and gallium compounds on ribonucleotide reductase

    SciTech Connect

    Narasimhan, J.

    1992-01-01

    The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied. The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy. Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits. M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites. M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme. In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols. Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content. Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown. The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit. The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven. The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds. Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit. The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system. The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit.

  6. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2.

    PubMed

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-09-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity. PMID:22895183

  7. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  8. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  9. Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1

    PubMed Central

    Yu, Xiaoliang; Li, Yun; Chen, Qin; Su, Chenhe; Zhang, Zili; Yang, Chengkui; Hu, Zhilin; Hou, Jue; Zhou, Jinying; Gong, Ling; Jiang, Xuejun

    2015-01-01

    ABSTRACT Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells. IMPORTANCE This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV. PMID:26559832

  10. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

  11. Diversity in Overall Activity Regulation of Ribonucleotide Reductase*

    PubMed Central

    Jonna, Venkateswara Rao; Crona, Mikael; Rofougaran, Reza; Lundin, Daniel; Johansson, Samuel; Brännström, Kristoffer; Sjöberg, Britt-Marie; Hofer, Anders

    2015-01-01

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives. PMID:25971975

  12. Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

    PubMed Central

    Roman, Nicholas O.; Samulitis, Betty K.; Wisner, Lee; Landowski, Terry H.; Dorr, Robert T.

    2010-01-01

    Purpose Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. Methods Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. Results In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. Conclusions These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer. PMID:20339847

  13. Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

    PubMed Central

    Heineman, T C; Cohen, J I

    1994-01-01

    Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir. Images PMID:8151792

  14. A phase II trial of sequential ribonucleotide reductase inhibition in aggressive myeloproliferative neoplasms

    PubMed Central

    Zeidner, Joshua F.; Karp, Judith E.; Blackford, Amanda L.; Smith, B. Douglas; Gojo, Ivana; Gore, Steven D.; Levis, Mark J.; Carraway, Hetty E.; Greer, Jacqueline M.; Ivy, S. Percy; Pratz, Keith W.; McDevitt, Michael A.

    2014-01-01

    Myeloproliferative neoplasms are a varied group of disorders that can have prolonged chronic phases, but eventually accelerate and can transform into a secondary acute myeloid leukemia that is ultimately fatal. Triapine is a novel inhibitor of the M2 subunit of ribonucleotide reductase. Sequential inhibition of ribonucleotide reductase with triapine and an M1 ribonucleotide reductase inhibitor (fludarabine) was noted to be safe, and led to a 29% complete plus partial response rate in myeloproliferative neoplasms. This article reports the findings of a phase II trial of triapine (105 mg/m2/day) followed by fludarabine (30 mg/m2/day) daily for 5 consecutive days in 37 patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. The overall response rate was 49% (18/37), with a complete remission rate of 24% (9/37). Overall response rates and complete remissions were seen in all disease subsets, including secondary acute myeloid leukemia, in which the overall response rate and complete remission rate were 48% and 33%, respectively. All patients with known JAK2 V617F mutations (6/6) responded. The median overall survival of the entire cohort was 6.9 months, with a median overall survival of both overall responders and complete responders of 10.6 months. These data further demonstrate the promise of sequential inhibition of ribonucleotide reductase in patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. This study was registered with clinicaltrials.gov (NCT00381550). PMID:24362550

  15. Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase.

    PubMed

    Szekeres, T; Gharehbaghi, K; Fritzer, M; Woody, M; Srivastava, A; van't Riet, B; Jayaram, H N; Elford, H L

    1994-01-01

    Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC50) at 5 microM, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC50 of 500 microM. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 microM, a concentration that inhibits cell proliferation by 50% (IC50) or at 100 microM for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC50 values of 7.5 and 50 microM, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC50 values of 12 and 87 microM, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82% in male mice and 112% in female mice). The anti-tumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans. PMID:8174204

  16. The Dimanganese(II) Site of Bacillus subtilis Class Ib Ribonucleotide Reductase

    SciTech Connect

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C.

    2014-10-02

    Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn{sub 2}{sup III}-Y{sm_bullet}, in their homodimeric NrdF ({beta}2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn{sub 2}{sup II} form of NrdF is an important component in understanding O{sub 2}-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn{sub 2}{sup II}-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 {angstrom} resolution crystal structure of Bacillus subtilis Mn{sub 2}{sup II}-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the {beta}2 dimer are distinct from those observed in E. coli Mn{sub 2}{sup II}-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn{sub 2}{sup II} cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly in class Ib RNRs.

  17. The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant

    PubMed Central

    Wei, Yifeng; Funk, Michael A.; Rosado, Leonardo A.; Baek, Jiyeon; Drennan, Catherine L.; Stubbe, JoAnne

    2014-01-01

    The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism. PMID:25157154

  18. Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

    PubMed Central

    Pizano, Arturo A.; Lutterman, Daniel A.; Holder, Patrick G.; Teets, Thomas S.; Stubbe, JoAnne; Nocera, Daniel G.

    2012-01-01

    Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state 3[Re ]∗ is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates. PMID:22171005

  19. Ribonucleotides

    PubMed Central

    Sutherland, John D.

    2010-01-01

    It has normally been assumed that ribonucleotides arose on the early Earth through a process in which ribose, the nucleobases, and phosphate became conjoined. However, under plausible prebiotic conditions, condensation of nucleobases with ribose to give β-ribonucleosides is fraught with difficulties. The reaction with purine nucleobases is low-yielding and the reaction with the canonical pyrimidine nucleobases does not work at all. The reasons for these difficulties are considered and an alternative high-yielding synthesis of pyrimidine nucleotides is discussed. Fitting the new synthesis to a plausible geochemical scenario is a remaining challenge but the prospects appear good. Discovery of an improved method of purine synthesis, and an efficient means of stringing activated nucleotides together, will provide underpinning support to those theories that posit a central role for RNA in the origins of life. PMID:20452951

  20. Mechanism of ribonucleotide reductase from Herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

    SciTech Connect

    Ator, M.A.; Stubbe, J.; Spector, T.

    1986-03-15

    Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of (3'-3H)ADP, (3'-H)UDP, and (5-3H) UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of (3'-3H)ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of (beta-32P)ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase.

  1. Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

    PubMed Central

    Rubin, H; Salem, J S; Li, L S; Yang, F D; Mama, S; Wang, Z M; Fisher, A; Hamann, C S; Cooperman, B S

    1993-01-01

    Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle. Images Fig. 2 Fig. 4 Fig. 5 PMID:8415692

  2. Determination of triapine, a ribonucleotide reductase inhibitor, in human plasma by liquid chromatography tandem mass spectrometry.

    PubMed

    Feng, Ye; Kunos, Charles A; Xu, Yan

    2015-09-01

    Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation-mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC-MS/MS method for the determination of triapine in human plasma. In this method, 2-[(3-fluoro-2-pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP18 column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mM, pH 8.50; v/v); column eluate was monitored by positive turbo-ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple-reaction-monitoring mode. The method developed had a linear calibration range of 0.250-50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS-normalized recovery and the IS-normalized matrix factor of triapine were 101-104% and 0.89-1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC-MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial. PMID:25677991

  3. Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

    PubMed Central

    Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

    2013-01-01

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

  4. Choosing the right metal: case studies of class I ribonucleotide reductases.

    PubMed

    Huang, Mingxia; Parker, Mackenzie J; Stubbe, JoAnne

    2014-10-10

    Over one-third of all proteins require metallation for function (Waldron, K. J., Rutherford, J. C., Ford, D., and Robinson, N.J. (2009) Nature 460, 823-830). As biochemical studies of most proteins depend on their isolation subsequent to recombinant expression (i.e. they are seldom purified from their host organism), there is no gold standard to assess faithful metallocofactor assembly and associated function. The biosynthetic machinery for metallocofactor formation in the recombinant expression system may be absent, inadequately expressed, or incompatible with a heterologously expressed protein. A combination of biochemical and genetic studies has led to the identification of key proteins involved in biosynthesis and likely repair of the metallocofactor of ribonucleotide reductases in both bacteria and the budding yeast. In this minireview, we will discuss the recent progress in understanding controlled delivery of metal, oxidants, and reducing equivalents for cofactor assembly in ribonucleotide reductases and highlight issues associated with controlling Fe/Mn metallation and avoidance of mismetallation. PMID:25160629

  5. Marek’s Disease Virus Encoded Ribonucleotide Reductase Large Subunit is not Essential for In Vitro Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) infected cells express a viral ribonucleotide reductase (RR) that is distinguishable from that present in uninfected cells by monoclonal antibody T81. Open reading frames UL39 and UL40 of the MDV genome encode the large (RR1) and small (RR2) subunits of RR enzyme, respe...

  6. Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses.

    PubMed

    Sakowski, Eric G; Munsell, Erik V; Hyatt, Mara; Kress, William; Williamson, Shannon J; Nasko, Daniel J; Polson, Shawn W; Wommack, K Eric

    2014-11-01

    Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O2 reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the "kill the winner-cost of resistance" model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations. PMID:25313075

  7. Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses

    PubMed Central

    Sakowski, Eric G.; Munsell, Erik V.; Hyatt, Mara; Kress, William; Williamson, Shannon J.; Nasko, Daniel J.; Polson, Shawn W.; Wommack, K. Eric

    2014-01-01

    Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O2 reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the “kill the winner–cost of resistance” model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations. PMID:25313075

  8. Identification and in vitro characterization of a Marek's disease virus-encoded Ribonucleotide reductase.

    PubMed

    Lee, Lucy F; Heidari, Mohammad; Sun, Aijun; Zhang, Huanmin; Lupiani, Blanca; Reddy, Sanjay

    2013-06-01

    Marek's disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acids whereas the small subunit encoded by UL40 (RR2) is predicted to be 343 amino acids long. Immunoprecipitation analysis of MDV-1 (GA strain)-infected cells with T81, a monoclonal antibody specific for RR of MDV, identified two major proteins of 90,000 and 40,000 daltons, corresponding to RR1 and RR2, respectively. In addition, RR was abundantly expressed in the cytoplasm of cells infected with 51 strains of MDV belonging to MDV serotypes 1, 2, and 3 as demonstrated by immunofluorescence staining. Northern blot analysis of RNA extracted from MDV-infected cells showed a major band of around 4.4 kb in size corresponding to the RR1 and RR2 transcripts. In vivo, RR was abundantly expressed in lymphoid organs and feather follicle epithelium of MDV-infected chickens during early cytolytic infection, as determined by immunohistochemistry. There was, however, no expression of RR in MDV-induced tumors in lymphoid organs. The abundant expression of RR in MDV-infected chicken may suggest an important role of RR in the conversion of ribonucleotides to deoxyribonucleotides for MDV DNA synthesis. PMID:24689171

  9. Marek’s disease virus encoded ribonucleotide reductase large subunit is essential for in vivo replication and plays a critical role in viral pathogenesis.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus encodes a ribonucleotide reductase (RR) that consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme and both subunits are necessary for enzyme activity. It is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleo...

  10. Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

    PubMed Central

    Wijerathna, Sanath R.; Ahmad, Md. Faiz; Xu, Hai; Fairman, James W.; Zhang, Andrew; Kaushal, Prem Singh; Wan, Qun; Kiser, Jianying; Dealwis, Chris G.

    2011-01-01

    Ribonucleotide reductase (RR) is a crucial enzyme in de novo DNA synthesis, where it catalyses the rate determining step of dNTP synthesis. RRs consist of a large subunit called RR1 (α), that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (β), which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an αnβm hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly. PMID:23115527

  11. Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme.

    PubMed

    Misko, Tessianna A; Wijerathna, Sanath R; Radivoyevitch, Tomas; Berdis, Anthony J; Ahmad, Md Faiz; Harris, Michael E; Dealwis, Chris G

    2016-06-01

    Sml1 is an intrinsically disordered protein inhibitor of Saccharomyces cerevisiae ribonucleotide reductase (ScRR1), but its inhibition mechanism is poorly understood. RR reduces ribonucleoside diphosphates to their deoxy forms, and balances the nucleotide pool. Multiple turnover kinetics show that Sml1 inhibition of dGTP/ADP- and ATP/CDP-bound ScRR follows a mixed inhibition mechanism. However, Sml1 cooperatively binds to the ES complex in the dGTP/ADP form, whereas with ATP/CDP, Sml1 binds weakly and noncooperatively. Gel filtration and mutagenesis studies indicate that Sml1 does not alter the oligomerization equilibrium and the CXXC motif is not involved in the inhibition. The data suggest that Sml1 is an allosteric inhibitor. PMID:27155231

  12. Large and small subunits of the Aujeszky's disease virus ribonucleotide reductase: nucleotide sequence and putative structure.

    PubMed

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-09-13

    We determined the entire DNA sequence of two adjacent open reading frames of Aujeszky's disease virus encoding ribonucleotide reductase genes with the intergenic sequence of 9 bp. From the sequence analysis we deduce that ORFs encode large and small subunits, with sizes of 835 and 303 amino acids, respectively. Amino acid sequence comparison of ADV RR2 with that of equine herpesvirus type 1, bovine herpesvirus type 1, HSV-1 and varicella zoster virus revealed that 48% of amino acids represent clusters of residues conserved in all compared sequences. In the N-terminal part ADV RR1 shows low homology to the RR1 of other herpesviruses. Rest of the RR1 protein contains highly conserved amino acid sequences divided by blocks of low homology. PMID:8086454

  13. Redox-dependent structural coupling between the α2 and β2 subunits in E. coli ribonucleotide reductase.

    PubMed

    Offenbacher, Adam R; Watson, R Atlee; Pagba, Cynthia V; Barry, Bridgette A

    2014-03-20

    Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2β2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in β2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2β2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and β2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2β2 and α2(13)Cβ2, were used to define subunit-specific structural changes. Mixing of α2 and β2 under turnover conditions yielded amide I (C═O) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to β strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as β2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, (2)H4 labeling of β2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated β2, were observed in the α2β2 complex. These spectra also provide evidence for a conformational rearrangement at an additional β2 tyrosine(s), Yx, in the α2β2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes. PMID:24606240

  14. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  15. Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers

    SciTech Connect

    Sommerhalter, M.; Voegtli, W.C.; Perlstein, D.L.; Ge, J.; Stubbe, J.; Rosenzweig, A.C.

    2010-03-08

    Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix {alpha}B, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices {alpha}A and {alpha}3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.

  16. Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate*

    PubMed Central

    Berggren, Gustav; Duraffourg, Nicolas; Sahlin, Margareta; Sjöberg, Britt-Marie

    2014-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and β subunits and are further divided into different subclasses, partly based on the metal content of the β-subunit. In subclass Ib RNR the β-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant. PMID:25262022

  17. RNA-dependent inhibition of ribonucleotide reductase is a major pathway for 5-azacytidine activity in acute myeloid leukemia

    PubMed Central

    Aimiuwu, Josephine; Wang, Hongyan; Chen, Ping; Xie, Zhiliang; Wang, Jiang; Liu, Shujun; Klisovic, Rebecca; Mims, Alice; Blum, William; Marcucci, Guido

    2012-01-01

    5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphosphate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucleotides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC–induced RRM2 gene expression inhibition involves its direct RNA incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination. PMID:22517893

  18. Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis * ♦

    PubMed Central

    Rhodes, DeLacy V.; Crump, Katie E.; Makhlynets, Olga; Snyder, Melanie; Ge, Xiuchun; Xu, Ping; Stubbe, JoAnne; Kitten, Todd

    2014-01-01

    Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259–6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y•) in vitro, whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor requires NrdI, and MnIII2-Y• is more active than FeIII2-Y• with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y•-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets. PMID:24381171

  19. Ribonucleotide reduction and the possible role of cobalamin in evolution.

    PubMed

    Dickman, S R

    1977-12-29

    The biological pathways of ribonucleotide reduction are briefly reviewed. The hypothesis is presented that reduction of ribonucleoside triphosphates to their deoxynucleotide analogs through the mediation of vitamin B12 or a similar corrinoid preceded and was necessary for the subsequent development of a DNA-type genome. There are two known biological systems for ribonucleotide reduction: (1) The ribonucleoside diphosphate reduction system which utilizes a nonheme iron ribonucleotide reductase enzyme, thioredoxin and its reductase, and NADPH. This enzyme complex is found in most bacteria, some higher organisms, and in all animals. (2) The ribonucleoside triphosphate reduction system which utilizes adenosyl cobalamin, ribonucleotide reductase and either thioredoxin or a disulfhydryl compound. The cobalamin-dependent reductase is restricted to a few species of bacteria and blue-gree algae. This system is considered more primitive than the iron reductase one based on their differences in distribution, components, and products. PMID:599575

  20. Autophagy induction causes a synthetic lethal sensitization to ribonucleotide reductase inhibition in breast cancer cells

    PubMed Central

    Chen, Yun-Ru; Tsou, Brittany; Hu, Shuya; Ma, Huimin; Liu, Xiyong; Yen, Yun; Ann, David K.

    2016-01-01

    Macroautophagy can promote cellular survival or death depending on the cellular context and its extent. We hypothesized that autophagy induction would synergize with a therapeutic agent targeting the autophagic cargo. To test this hypothesis, we treated breast cancer MDA-MB-231 cells with tamoxifen (TMX), which induces autophagy through an estrogen receptor-independent pathway. Induction of autophagy reduced cellular levels of RRM2, a subunit of ribonucleotide reductase (RR), the rate limiting enzyme in the production of deoxyribonucleotide triphosphates (dNTPs). This autophagy inducer was combined with COH29, an inhibitor developed in our laboratory that targets RR through a novel mechanism. The combination therapy showed synergistic effects on cytotoxicity in vitro and in an in vivo xenograft model. This cytotoxicity was blocked by knockdown of the autophagy protein ATG5 or addition of chloroquine, an autophagy inhibitor. The combined therapy also induced dNTP depletion and massive genomic instability, leading us to hypothesize that combining autophagy induction with RR inhibition can lead to mitotic catastrophe in rapidly dividing cells. We propose that this TMX + COH29 combined therapy may have clinical benefit. Furthermore, autophagy induction may be a general mechanism for augmenting the effects of chemotherapeutic agents PMID:26675256

  1. Deciphering Radical Transport in the Catalytic Subunit of Class I Ribonucleotide Reductase

    PubMed Central

    Holder, Patrick G.; Pizano, Arturo A.; Anderson, Bryce L.; Stubbe, JoAnne; Nocera, Daniel G.

    2012-01-01

    Incorporation of 2,3,6-trifluorotyrosine (F3Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F3Y radical from the [Re]–F3Y–βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y–Y established, radical transport occurs with a rate constant of 3 × 105 s−1. Point mutations that disrupt the Y–Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the co-linear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme. PMID:22121977

  2. A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

    PubMed

    Fu, Yuan; Lin, Hong-Yu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

    2014-11-24

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation. PMID:25256246

  3. Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Inhibitor Sml1 in Response to Iron Deficiency

    PubMed Central

    Sanvisens, Nerea; Romero, Antonia M.; An, Xiuxiang; Zhang, Caiguo; de Llanos, Rosa; Martínez-Pastor, María Teresa; Bañó, M. Carmen

    2014-01-01

    Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox-active cofactor in many biological processes, including DNA replication and repair. Eukaryotic ribonucleotide reductases (RNRs) are Fe-dependent enzymes that catalyze deoxyribonucleoside diphosphate (dNDP) synthesis. We show here that the levels of the Sml1 protein, a yeast RNR large-subunit inhibitor, specifically decrease in response to both nutritional and genetic Fe deficiencies in a Dun1-dependent but Mec1/Rad53- and Aft1-independent manner. The decline of Sml1 protein levels upon Fe starvation depends on Dun1 forkhead-associated and kinase domains, the 26S proteasome, and the vacuolar proteolytic pathway. Depletion of core components of the mitochondrial iron-sulfur cluster assembly leads to a Dun1-dependent diminution of Sml1 protein levels. The physiological relevance of Sml1 downregulation by Dun1 under low-Fe conditions is highlighted by the synthetic growth defect observed between dun1Δ and fet3Δ fet4Δ mutants, which is rescued by SML1 deletion. Consistent with an increase in RNR function, Rnr1 protein levels are upregulated upon Fe deficiency. Finally, dun1Δ mutants display defects in deoxyribonucleoside triphosphate (dNTP) biosynthesis under low-Fe conditions. Taken together, these results reveal that the Dun1 checkpoint kinase promotes RNR function in response to Fe starvation by stimulating Sml1 protein degradation. PMID:24958100

  4. Herpes simplex virus specifies two subunits of ribonucleotide reductase encoded by 3'-coterminal transcripts.

    PubMed Central

    Swain, M A; Galloway, D A

    1986-01-01

    We have previously described a transcription unit located between map coordinates 0.558 and 0.595 on the herpes simplex virus type 2 strain 333 genome which encodes two mRNAs of 5.0 and 1.2 kilobases that share a common 3' terminus, and we have determined the nucleotide sequence of a 38,000-dalton protein specified by the smaller RNA (D. A. Galloway and M. A. Swain, J. Virol. 49:724-730, 1984). The entire nucleotide sequence of the 140,000-dalton protein specified by a 3,432-base-pair open reading frame within the large mRNA is presented, as are transcriptional regulatory sequences upstream of the RNA. The 140,000-dalton protein shows strong homology with the large subunit of well-characterized ribonucleotide reductase enzymes from the mouse and from Escherichia coli and with an Epstein-Barr virus gene. The 38,000-dalton protein has been shown previously to have homology with the small subunit of these enzymes (B.-M. Sjoberg, H. Eklund, J. A. Fuchs, J. Carlson, N. M. Standart, J. V. Ruderman, S. J. Bray, and T. Hunt, FEBS Lett. 183:99-102, 1985). This is the first example of a herpesvirus transcriptional unit that encodes functionally related proteins. PMID:2419588

  5. The Structural Basis for Peptidomimetic Inhibition of Eukaryotic Ribonucleotide Reductase: A Conformationally Flexible Pharmacophore

    SciTech Connect

    Xu, Hai; Fairman, James W.; Wijerathna, Sanath R.; Kreischer, Nathan R.; LaMacchia, John; Helmbrecht, Elizabeth; Cooperman, Barry S.; Dealwis, Chris

    2008-08-19

    Eukaryotic ribonucleotide reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is a target for cancer therapy. RR activity requires formation of a complex between subunits R1 and R2 in which the R2 C-terminal peptide binds to R1. Here we report crystal structures of heterocomplexes containing mammalian R2 C-terminal heptapeptide, P7 (Ac-{sup 1}FTLDADF{sup 7}) and its peptidomimetic P6 ({sup 1}Fmoc(Me)PhgLDChaDF{sup 7}) bound to Saccharomyces cerevisiae R1 (ScR1). P7 and P6, both of which inhibit ScRR, each bind at two contiguous sites containing residues that are highly conserved among eukaryotes. Such binding is quite distinct from that reported for prokaryotes. The Fmoc group in P6 peptide makes several hydrophobic interactions that contribute to its enhanced potency in binding to ScR1. Combining all of our results, we observe three distinct conformations for peptide binding to ScR1. These structures provide pharmacophores for designing highly potent nonpeptide class I RR inhibitors.

  6. Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

    SciTech Connect

    Zimanyi, Christina M.; Ando, Nozomi; Brignole, Edward J.; Asturias, Francisco J.; Stubbe, JoAnne; Drennan, Catherine L.

    2012-10-23

    Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into {alpha}{sub 4}{beta}{sub 4} rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described {alpha}{sub 4}{beta}{sub 4} rings can interlock to form an unprecedented ({alpha}{sub 4}{beta}{sub 4}){sub 2} megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

  7. Shift in Ribonucleotide Reductase Gene Expression in Pseudomonas aeruginosa during Infection ▿ †

    PubMed Central

    Sjöberg, Britt-Marie; Torrents, Eduard

    2011-01-01

    The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients. PMID:21502590

  8. Tangled up in knots – Structures of inactivated forms of E. coli class Ia Ribonucleotide Reductase

    PubMed Central

    Zimanyi, Christina M.; Ando, Nozomi; Brignole, Edward J.; Asturias, Francisco J.; Stubbe, JoAnne; Drennan, Catherine L.

    2012-01-01

    Summary Successful targets for anti-cancer drugs such as clofarabine and gemcitabine, ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNR as well as present a mechanism for the formation of these unusual structures. PMID:22727814

  9. Structural Basis for Allosteric Regulation of Human Ribonucleotide Reductase by Nucleotide-induced Oligomerization

    SciTech Connect

    J Fairman; S Wijerathna; M Ahmad; H Xu; R nakano; S jha; J Prendergast; R Welin; S Flodin; et al.

    2011-12-31

    Ribonucleotide reductase (RR) is an {alpha}{sub n}{beta}{sub n} (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the {alpha}{sub 6}-{beta}{beta}'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

  10. Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo

    PubMed Central

    Cotruvo, Joseph A.; Stubbe, JoAnne

    2015-01-01

    Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an FeIIIFeIII-tyrosyl radical (Y•) cofactor (class Ia), a MnIIIMnIII-Y• cofactor (class Ib), and a MnIVFeIII cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our under-standing of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields. PMID:21456967

  11. An assay for ribonucleotide reductase based on ion-exchange chromatography of the reaction product.

    PubMed

    Narine, D R; Bacchetti, S; Chan, W W

    1985-03-01

    A rapid and convenient assay for ribonucleotide reductase has been developed in which the reaction product, deoxycytidine diphosphate (dCDP), is isolated without further conversion. The enzymatic reaction is terminated by the addition of ethanol and the sample is chromatographed on a single, small, and disposable column of polyethylenimine cellulose. A two-step elution is conducted with buffers containing 25% ethanol. First, contaminants and byproducts such as cytidine and its monophosphate are removed at low ionic strength while the diphosphates are retained. Then dCDP is selectively eluted as a sharp peak with a strong borate buffer. Under these conditions, the excess substrate, cytidine diphosphate, remains on the column, presumably as the borate complex. The assay is linear with time for 15 min at 25 degrees C and linear with the amount of enzyme even at very low concentrations. With slight modifications, the assay seems applicable to the use of UDP or ADP as substrates. The method is not suitable for samples which contain nucleotide kinase or other interfering enzymes which convert a significant amount of dCDP into byproducts. However, another chromatographic system based on similar principles has been found which could be used to measure any dCTP produced in this way. PMID:2990252

  12. A bioinformatic analysis of ribonucleotide reductase genes in phage genomes and metagenomes

    PubMed Central

    2013-01-01

    Background Ribonucleotide reductase (RNR), the enzyme responsible for the formation of deoxyribonucleotides from ribonucleotides, is found in all domains of life and many viral genomes. RNRs are also amongst the most abundant genes identified in environmental metagenomes. This study focused on understanding the distribution, diversity, and evolution of RNRs in phages (viruses that infect bacteria). Hidden Markov Model profiles were used to analyze the proteins encoded by 685 completely sequenced double-stranded DNA phages and 22 environmental viral metagenomes to identify RNR homologs in cultured phages and uncultured viral communities, respectively. Results RNRs were identified in 128 phage genomes, nearly tripling the number of phages known to encode RNRs. Class I RNR was the most common RNR class observed in phages (70%), followed by class II (29%) and class III (28%). Twenty-eight percent of the phages contained genes belonging to multiple RNR classes. RNR class distribution varied according to phage type, isolation environment, and the host’s ability to utilize oxygen. The majority of the phages containing RNRs are Myoviridae (65%), followed by Siphoviridae (30%) and Podoviridae (3%). The phylogeny and genomic organization of phage and host RNRs reveal several distinct evolutionary scenarios involving horizontal gene transfer, co-evolution, and differential selection pressure. Several putative split RNR genes interrupted by self-splicing introns or inteins were identified, providing further evidence for the role of frequent genetic exchange. Finally, viral metagenomic data indicate that RNRs are prevalent and highly dynamic in uncultured viral communities, necessitating future research to determine the environmental conditions under which RNRs provide a selective advantage. Conclusions This comprehensive study describes the distribution, diversity, and evolution of RNRs in phage genomes and environmental viral metagenomes. The distinct distributions of

  13. Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide

    SciTech Connect

    Chen, Zhengming; Zhou, Jun; Zhang, Yingtao; Bepler, Gerold

    2011-09-23

    Highlights: {yields} Gemcitabine induces a RRM1 conformational change in tumor cell lines and xenografts. {yields} The 110 kDa RRM1 is unique to gemcitabine interaction among 12 cytotoxic agents. {yields} The 110 kDa RRM1 can be stabilized by the thiol alkylator N-ethylmaleimide. {yields} C218A, C429A, and E431A mutations in RRM1 abolished the conformational change. {yields} The 110 kDa RRM1 may be a specific biomarker of gemcitabine's therapeutic efficacy. -- Abstract: Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2',2'-difluoro-2'-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110 kDa band, along with the 90 kDa native RRM1 band, appeared in immunoblots. This 110 kDa band was identified as RRM1 by mass spectrometry (LC-MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. It is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to

  14. A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair.

    PubMed

    Fujii, Naoaki; Evison, Benjamin J; Actis, Marcelo L; Inoue, Akira

    2015-11-01

    Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. PMID:26462050

  15. Ribonucleotide reductase small subunit M2B prognoses better survival in colorectal cancer

    PubMed Central

    Liu, Xiyong; Lai, Lily; Wang, Xiaochen; Xue, Lijun; Leora, Sofia; Wu, Jun; Hu, Shuya; Zhang, Keqiang; Kuo, Mei-Ling; Zhou, Lun; Zhang, Hang; Wang, Yafan; Wang, Yan; Zhou, Bingsen; Nelson, Rebecca A; Zheng, Shu; Zhang, Suzhan; Chu, Peiguo; Yen, Yun

    2011-01-01

    Ribonucleotide reductase subunit RRM2B (p53R2) has been reported to suppress invasion and metastasis in colorectal cancer (CRC). Here we report that high levels of RRM2B expression is correlated with markedly better survival in CRC patients. In a fluorescence-labeled orthotopic mouse xenograft model, we confirmed that overexpression of RRM2B in non-metastatic CRC cells prevented lung and/or liver metastasis, relative to control cells that did metastasize. Clinical outcome studies were conducted on a training set with 103 CRCs and a validation set with 220 CRCs. All participants underwent surgery with periodic follow-up to determine survivability. A newly developed specific RRM2B antibody was employed to perform immunohistochemistry (IHC) for determining RRM2B expression levels on tissue arrays. In the training set, the Kaplan-Meier and multivariate COX analysis revealed that RRM2B is associated with better survival of CRCs, especially in stage IV patients (Hazard ratio, HR=0.40; 95% CI 0.18–0.86, p=0.016). In the validation set, RRM2B was negatively related to tumor invasion (odds ratio, OR=0.45, 95% CI 0.19–0.99, p=0.040) and lymph node involvement (OR=0.48, 95% CI 0.25–0.92, p=0.026). Further, elevated expression of RRM2B was associated with better prognosis in this set as determined by multivariate analyses (HR=0.48, 95% CI 0.26–0.91, p=0.030). Further investigations revealed that RRM2B was correlated with better survival of CRCs with advanced stage III–IV tumors rather than earlier stage I–II tumors. Taken together, our findings establish that RRM2B suppresses invasiveness of cancer cells and that its expression is associated with a better survival prognosis for CRC patients. PMID:21415168

  16. Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein.

    PubMed

    Liu, Xia; Xu, Zhijian; Hou, Chuanwei; Wang, Meng; Chen, Xinhuan; Lin, Qinghui; Song, Rui; Lou, Meng; Zhu, Lijun; Qiu, Yunqing; Chen, Zhi; Yang, Chunhao; Zhu, Weiliang; Shao, Jimin

    2016-03-01

    Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment. PMID:26774458

  17. Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

    PubMed

    Rawson, Jonathan M O; Roth, Megan E; Xie, Jiashu; Daly, Michele B; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-06-01

    Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity. PMID:27117260

  18. The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro.

    PubMed

    Chen, Mei-Chuan; Zhou, Bingsen; Zhang, Keqiang; Yuan, Yate-Ching; Un, Frank; Hu, Shuya; Chou, Chih-Ming; Chen, Chun-Han; Wu, Jun; Wang, Yan; Liu, Xiyong; Smith, D Lynne; Li, Hongzhi; Liu, Zheng; Warden, Charles D; Su, Leila; Malkas, Linda H; Chung, Young Min; Hu, Mickey C-T; Yen, Yun

    2015-06-01

    COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1-defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5- and DR-green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent. PMID:25814515

  19. A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase

    PubMed Central

    Yokoyama, Kenichi; Uhlin, Ulla; Stubbe, JoAnne

    2010-01-01

    Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•) cofactor to initiate catalysis. The initiation process requires long range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122• → W48 → Y356 within β to Y731 → Y730 → C439 within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO2Y) has recently been incorporated site-specifically in place of Y122 in β2. The protein as isolated contained a diferric cluster, but no nitrotyrosyl radical (NO2Y•) and was inactive. In the present paper we show that incubation of apo-Y122NO2Y-β2 with Fe2+ and O2 generates a diferric-NO2Y• that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO2Y•/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped flow spectroscopy, rapid freeze quench EPR spectroscopy and rapid chemical quench methods. These studies have for the first time unmasked the conformational gating. They reveal that the NO2Y• is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s-1), that dCDP is produced at 107 s-1, and that a new Y• is produced at 97 s-1. Studies with pathway mutants suggest that the new Y• is predominantly located at 356 in β2. In conjunction with the crystal structure of Y122NO2Y-β2, a mechanism for PCET uncoupling in NO2Y•-RNR is proposed. PMID:20929229

  20. The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro

    PubMed Central

    Chen, Mei-Chuan; Zhou, Bingsen; Zhang, Keqiang; Yuan, Yate-Ching; Un, Frank; Hu, Shuya; Chou, Chih-Ming; Chen, Chun-Han; Wu, Jun; Wang, Yan; Liu, Xiyong; Smith, D. Lynne; Li, Hongzhi; Liu, Zheng; Warden, Charles D.; Su, Leila; Malkas, Linda H.; Chung, Young Min; Hu, Mickey C.-T.

    2015-01-01

    COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1–defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5– and DR–green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent. PMID:25814515

  1. Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis. PMID:25353063

  2. Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.

    PubMed

    Zhang, Yan; Stubbe, Joanne

    2011-06-28

    Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)(2)-Y(•)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/β(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(•) [Mn(III)(2)-Y(•)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(•) and Fe(III)(2)-Y(•) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(•)/β(2)). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)(2)-Y(•) center with 2 Mn/β(2) and 0.5 Y(•)/β(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(•)/β(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(•) enzyme. PMID:21561096

  3. A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase.

    PubMed

    Yokoyama, Kenichi; Uhlin, Ulla; Stubbe, JoAnne

    2010-11-01

    Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(•)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(•)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(•) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(•)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(•) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(•) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(•) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(•)-RNR is proposed. PMID:20929229

  4. Implications and problems in analysing cytotoxic activity of hydroxyurea in combination with a potential inhibitor of ribonucleotide reductase.

    PubMed

    Nocentini, G; Barzi, A; Franchetti, P

    1990-01-01

    The cytotoxicity of hydroxyurea in combination with 2.2'-bipyridyl-6-carbothioamide (a potential inhibitor of ribonucleotide reductase) on P388 murine leukemia is reported. Synergistic activity was studied using various interpretations of the isobologram method and the combination index method. We evaluated the pros and cons of these methods and their overall usefulness. In our opinion, to obtain all possible information from a compound association, it is important to choose a formally correct method that (a) can quantitatively evaluate synergism or antagonism, (b) may offer the possibility of averaging final results, (c) needs a minimal amount of experimental data, and (d) is rapid. Moreover, we emphasize both the utility of testing at least three molar ratios of compound association and the importance of carefully choosing the fractional inhibition used in calculating the combination effect. Such evaluation of drug combinations gives information essential to the preparation of new anticancer drug regimens and to the early assessment of biochemical interactions. PMID:2208576

  5. The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2*

    PubMed Central

    Chen, Xinhuan; Xu, Zhijian; Zhang, Lingna; Liu, Hongchuan; Liu, Xia; Lou, Meng; Zhu, Lijun; Huang, Bingding; Yang, Cai-Guang; Zhu, Weiliang; Shao, Jimin

    2014-01-01

    Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells. PMID:24253041

  6. Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

    PubMed Central

    Zhang, Zhen; An, Xiuxiang; Yang, Kui; Perlstein, Deborah L.; Hicks, Leslie; Kelleher, Neil; Stubbe, JoAnne; Huang, Mingxia

    2006-01-01

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (α, oligomeric state unknown) and a small subunit R2 (β2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe)2-Y·] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (ββ′), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ββ′ is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ββ′ nuclear localization: the importin β homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ββ′ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ββ′. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ββ′ by Kap122 or as an anchor to retain ββ′ in the nucleus. PMID:16432237

  7. Studies of Ribonucleotide Reductase in Crucian Carp—An Oxygen Dependent Enzyme in an Anoxia Tolerant Vertebrate

    PubMed Central

    Sandvik, Guro K.; Tomter, Ane B.; Bergan, Jonas; Zoppellaro, Giorgio; Barra, Anne-Laure; Røhr, Åsmund K.; Kolberg, Matthias; Ellefsen, Stian

    2012-01-01

    The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O2) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp. PMID:22916159

  8. Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

    PubMed Central

    Tomter, Ane B.; Zoppellaro, Giorgio; Bell, Caleb B.; Barra, Anne-Laure; Andersen, Niels H.; Solomon, Edward I.; Andersson, K. Kristoffer

    2012-01-01

    Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher

  9. 14-3-3σ regulation of and interaction with YAP1 in acquired gemcitabine resistance via promoting ribonucleotide reductase expression

    PubMed Central

    Qin, Li; Dong, Zizheng; Zhang, Jian-Ting

    2016-01-01

    Gemcitabine is an important anticancer therapeutics approved for treatment of several human cancers including locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). Its clinical effectiveness, however, is hindered by existence of intrinsic and development of acquired resistances. Previously, it was found that 14-3-3σ expression associates with poor clinical outcome of PDAC patients. It was also found that 14-3-3σ expression is up-regulated in gemcitabine resistant PDAC cells and contributes to the acquired gemcitabine resistance. In this study, we investigated the molecular mechanism of 14-3-3σ function in gemcitabine resistance and found that 14-3-3σ up-regulates YAP1 expression and then binds to YAP1 to inhibit gemcitabine-induced caspase 8 activation and apoptosis. 14-3-3σ association with YAP1 up-regulates the expression of ribonucleotide reductase M1 and M2, which may mediate 14-3-3σ/YAP1 function in the acquired gemcitabine resistance. These findings suggest a possible role of YAP1 signaling in gemcitabine resistance. PMID:26894857

  10. Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.

    PubMed

    Artin, Erin; Wang, Jun; Lohman, Gregory J S; Yokoyama, Kenichi; Yu, Guixue; Griffin, Robert G; Bar, Galit; Stubbe, JoAnne

    2009-12-15

    Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv of F(2)CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of alpha with the sugar of F(2)CDP (one label/alpha2beta2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that alpha migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-alpha in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-alpha migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1'-(2)H]-F(2)CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of g(x) 2.00738, g(y) 2.00592, and g(z) 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1'-(1)H]-F(2)CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR. PMID:19899770

  11. A stable Fe{sup III}-Fe{sup IV} replacement of tyrosyl radical in a class I ribonucleotide reductase

    SciTech Connect

    Voevodskaya, N.; Lendzian, F.; Graeslund, A. . E-mail: astrid@dbb.su.se

    2005-05-20

    Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe{sup III}-Fe{sup IV} species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.

  12. DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2016-02-01

    Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage. PMID:26247322

  13. A tyrosine–tryptophan dyad and radical-based charge transfer in a ribonucleotide reductase-inspired maquette

    PubMed Central

    Pagba, Cynthia V.; McCaslin, Tyler G.; Veglia, Gianluigi; Porcelli, Fernando; Yohannan, Jiby; Guo, Zhanjun; McDaniel, Miranda; Barry, Bridgette A.

    2015-01-01

    In class 1a ribonucleotide reductase (RNR), a substrate-based radical is generated in the α2 subunit by long-distance electron transfer involving an essential tyrosyl radical (Y122O·) in the β2 subunit. The conserved W48 β2 is ∼10 Å from Y122OH; mutations at W48 inactivate RNR. Here, we design a beta hairpin peptide, which contains such an interacting tyrosine–tryptophan dyad. The NMR structure of the peptide establishes that there is no direct hydrogen bond between the phenol and the indole rings. However, electronic coupling between the tyrosine and tryptophan occurs in the peptide. In addition, downshifted ultraviolet resonance Raman (UVRR) frequencies are observed for the radical state, reproducing spectral downshifts observed for β2. The frequency downshifts of the ring and CO bands are consistent with charge transfer from YO· to W or another residue. Such a charge transfer mechanism implies a role for the β2 Y-W dyad in electron transfer. PMID:26627888

  14. Radiosensitization of Human Cervical Cancer Cells by Inhibiting Ribonucleotide Reductase: Enhanced Radiation Response at Low-Dose Rates

    SciTech Connect

    Kunos, Charles A.; Colussi, Valdir C.; Pink, John; Radivoyevitch, Tomas; Oleinick, Nancy L.

    2011-07-15

    Purpose: To test whether pharmacologic inhibition of ribonucleotide reductase (RNR) by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC no. 663249) enhances radiation sensitivity during low-dose-rate ionizing radiation provided by a novel purpose-built iridium-192 cell irradiator. Methods and Materials: The cells were exposed to low-dose-rate radiation (11, 23, 37, 67 cGy/h) using a custom-fabricated cell irradiator or to high-dose-rate radiation (330 cGy/min) using a conventional cell irradiator. The radiation sensitivity of human cervical (CaSki, C33-a) cancer cells with or without RNR inhibition by 3-AP was evaluated using a clonogenic survival and an RNR activity assay. Alteration in the cell cycle distribution was monitored using flow cytometry. Results: Increasing radiation sensitivity of both CaSki and C33-a cells was observed with the incremental increase in radiation dose rates. 3-AP treatment led to enhanced radiation sensitivity in both cell lines, eliminating differences in cell cytotoxicity from the radiation dose rate. RNR blockade by 3-AP during low-dose-rate irradiation was associated with low RNR activity and extended G{sub 1}-phase cell cycle arrest. Conclusions: We conclude that RNR inhibition by 3-AP impedes DNA damage repair mechanisms that rely on deoxyribonucleotide production and thereby increases radiation sensitivity of human cervical cancers to low-dose-rate radiation.

  15. Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides

    PubMed Central

    Dang, Thao P.; Sobczak, Adam J.; Mebel, Alexander M.; Chatgilialoglu, Chryssostomos; Wnuk, Stanislaw F.

    2012-01-01

    Model 3′-azido-3′-deoxynucleosides with thiol or vicinal dithiol substituents at C2′ or C5′ were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides. Esterification of 5′-(tert-butyldiphenylsilyl)-3′-azido-3′-deoxyadenosine and 3′-azido-3′-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3′-azido-3′-deoxy-2′-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6-41.2 kcal/mol and have low energy barriers of 10.4-13.5 kcal/mol. Reduction of the azido group occurred to give 3′-amino-3′-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl)thymidine with 2,2′-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N2O-saturated aqueous solutions of AZT and cysteine produced 3′-amino-3′-deoxythymidine and thymine most likely by both radical and ionic processes. PMID:22711937

  16. Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

    PubMed Central

    Cooper, J; Conner, J; Clements, J B

    1995-01-01

    We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases. PMID:7609068

  17. Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells.

    PubMed

    Pontarin, Giovanna; Ferraro, Paola; Bee, Leonardo; Reichard, Peter; Bianchi, Vera

    2012-08-14

    In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage. PMID:22847445

  18. Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

    PubMed Central

    Dreux, Nicolas; Cendra, Maria del Mar; Massier, Sébastien; Darfeuille-Michaud, Arlette

    2015-01-01

    A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib, and III), which are transcribed from separate operons and encoded by the genes nrdAB, nrdHIEF, and nrdDG, respectively. Here, we investigated the role of RNRs in the virulence of adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib, and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encode a transcriptional regulator of RNRs and class III anaerobic RNR, respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients. PMID:25605769

  19. Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase.

    PubMed

    Doan, Peter E; Shanmugam, Muralidharan; Stubbe, JoAnne; Hoffman, Brian M

    2015-12-16

    Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238. PMID:26636616

  20. Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study

    PubMed Central

    Popović-Bijelić, Ana; Kowol, Christian R.; Lind, Maria E.S.; Luo, Jinghui; Himo, Fahmi; Enyedy, Éva A.; Arion, Vladimir B.; Gräslund, Astrid

    2012-01-01

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper (II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron (II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical. PMID:21955844

  1. Direct Interfacial Y731 Oxidation in α2 by a Photoβ2 Subunit of E. coli Class Ia Ribonucleotide Reductase

    PubMed Central

    Song, David Y.; Pizano, Arturo A.; Holder, Patrick G.; Stubbe, JoAnne; Nocera, Daniel G.

    2015-01-01

    Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β2 and α2) involving multiple PCET steps and the conserved tyrosine triad [Y356(β2)–Y731(α2)–Y730(α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y356C-(β2) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F3Y) is incorporated in place of Y731 in α2. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re356]-β2:2,3,5-F3Y731-α2 in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α2 from β2 in the absence of the interfacial Y356 residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y731(α2), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356 and Y731, at the subunit interface. PMID:26504513

  2. Autocatalytic generation of dopa in the engineered protein R2 F208Y from Escherichia coli ribonucleotide reductase and crystal structure of the dopa-208 protein.

    PubMed

    Aberg, A; Ormö, M; Nordlund, P; Sjöberg, B M

    1993-09-21

    The mutant form Phe-208-->Tyr of the R2 protein of Escherichia coli ribonucleotide reductase contains an intrinsic ferric-Dopa cofactor with characteristic absorption bands at 460 and ca. 700 nm [Ormö, M., de Maré, F., Regnström, K., Aberg, A., Sahlin, M., Ling, J., Loehr, T. M., Sanders-Loehr, J., & Sjöberg, B. M. (1992) J. Biol. Chem. 267, 8711-8714]. The three-dimensional structure of the mutant protein, solved to 2.5-A resolution, shows that the Dopa is localized to residue 208 and that it is a bidentate ligand of Fe1 of the binuclear iron center of protein R2. Nascent apoR2 F208Y, lacking metal ions, can be purified from overproducing cells grown in iron-depleted medium. ApoR2 F208Y is rapidly and quantitatively converted to the Dopa-208 form in vitro by addition of ferrous iron in the presence of oxygen. Other metal ions (Cu2+, Mn2+, Co2+) known to bind to the metal site of wild-type apoR2 do not generate a Dopa in apoR2 F208Y. The autocatalytic generation of Dopa does not require the presence of a tyrosine residue at position 122, the tyrosine which in a wild-type R2 protein acquires the catalytically essential tyrosyl radical. It is proposed that generation of Dopa initially follows the suggested reaction mechanism for tyrosyl radical generation in the wild-type protein and involves a ferryl intermediate, which in the case of the mutant R2 protein oxygenates Tyr 208. This autocatalytic metal-mediated reaction in the engineered R2 F208Y protein may serve as a model for formation of covalently bound quinones in other proteins. PMID:8373782

  3. RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank

    PubMed Central

    2009-01-01

    Background Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes. Description In RNRdb (Ribonucleotide Reductase database), we have collated and curated all known RNR protein sequences with the aim of providing a resource for exploration of RNR diversity and distribution. By comparing expert manual annotations with annotations stored in Genbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23% of protein sequences included in RNRdb are correctly annotated across the key attributes of class, role and function, with 17% being incorrectly annotated across all three categories. This illustrates the utility of specialist databases for applications where a high degree of annotation accuracy may be important. The database houses information on annotation, distribution and diversity of RNRs, and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb is accessible through a public web interface at http://rnrdb.molbio.su.se. Conclusion RNRdb is a specialist database that provides a reliable annotation and classification resource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. The recent expansion in available genome sequence data have provided us with a picture of RNR distribution that is more complex than believed only a few years ago; our database indicates that RNRs of all three classes are found across all three cellular domains. Moreover, we find a number of organisms that

  4. Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors.

    PubMed

    Minami, Kentaro; Shinsato, Yoshinari; Yamamoto, Masatatsu; Takahashi, Homare; Zhang, Shaoxuan; Nishizawa, Yukihiko; Tabata, Sho; Ikeda, Ryuji; Kawahara, Kohich; Tsujikawa, Kazutake; Chijiiwa, Kazuo; Yamada, Katsushi; Akiyama, Shin-ichi; Pérez-Torras, Sandra; Pastor-Anglada, Marcal; Furukawa, Tatsuhiko; Yasuo, Takeda

    2015-03-01

    Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors. PMID:25837929

  5. Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

    PubMed

    Abbouni, Bouziane; Elhariry, Hesham M; Auling, Georg

    2003-01-01

    Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain. PMID:12882290

  6. Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

    SciTech Connect

    Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun; Wijerathna, Sanath R.; An, Xiuxiang; Huang, Mingxia; Dealwis, Chris Godfrey

    2012-11-01

    Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

  7. Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example

    PubMed Central

    2016-01-01

    Fluorinated tyrosines (FnY’s, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y’s, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y’s (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentials (−30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry. PMID:27276098

  8. A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

    PubMed Central

    Habtemichael, Negusse; Bier, Carolin; Unruhe, Britta; Weisheit, Simona; Spange, Stephanie; Nonnenmacher, Frank; Fetz, Verena; Ginter, Torsten; Reichardt, Sigrid; Liebmann, Claus; Schneider, Günter; Krämer, Oliver H.

    2012-01-01

    Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p<0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P<0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti

  9. Two distinct mechanisms of inactivation of the class Ic ribonucleotide reductase from Chlamydia trachomatis by hydroxyurea: implications for the protein gating of intersubunit electron transfer.

    PubMed

    Jiang, Wei; Xie, Jiajia; Varano, Paul T; Krebs, Carsten; Bollinger, J Martin

    2010-06-29

    Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the alpha(2) subunit is oxidized to a thiyl radical (C(*)) by a cofactor approximately 35 A away in the beta(2) subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y(*)) is the C oxidant, whereas a Mn(IV)/Fe(III) cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient "pathway" Y(*)s in a multistep electron transfer (ET) process that is "gated" by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic beta(2) subunits by reducing their C oxidants. Reduction of the stable cofactor Y(*) (Y122(*)) in Escherichia coli class Ia beta(2) is faster in the presence of alpha(2) and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y(*) under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the Mn(IV)/Fe(III) cofactor to give two distinct products: the previously described homogeneous Mn(III)/Fe(III)-beta(2) complex, which forms only under turnover conditions (in the presence of alpha(2) and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms approximately 900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of Mn(IV)/Fe(III)-beta(2) only. Formation of Mn(III)/Fe(III)-beta(2) also requires (i) either Y338, the subunit-interfacial ET pathway residue of beta(2), or Y222, the surface residue that relays the "extra electron" to the Mn(IV)/Fe(IV) intermediate during activation of beta(2) but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor

  10. Ribonucleotide Reductase Subunit M2 Predicts Survival in Subgroups of Patients with Non-Small Cell Lung Carcinoma: Effects of Gender and Smoking Status

    PubMed Central

    Mah, Vei; Alavi, Mohammad; Márquez-Garbán, Diana C.; Maresh, Erin L.; Kim, Sara R.; Horvath, Steve; Bagryanova, Lora; Huerta-Yepez, Sara; Chia, David; Pietras, Richard

    2015-01-01

    Background Ribonucleotide reductase catalyzes the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The functional enzyme consists of two subunits - one large (RRM1) and one small (RRM2 or RRM2b) subunit. Expression levels of each subunit have been implicated in prognostic outcomes in several different types of cancers. Experimental Design Immunohistochemistry for RRM1 and RRM2 was performed on a lung cancer tissue microarray (TMA) and analyzed. 326 patients from the microarray were included in this study. Results In non-small cell lung cancer (NSCLC), RRM2 expression was strongly predictive of disease-specific survival in women, non-smokers and former smokers who had quit at least 10 years prior to being diagnosed with lung cancer. Higher expression was associated with worse survival. This was not the case for men, current smokers and those who had stopped smoking for shorter periods of time. RRM1 was not predictive of survival outcomes in any subset of the patient group. Conclusion RRM2, but not RRM1, is a useful predictor of survival outcome in certain subsets of NSCLC patients. PMID:26001082

  11. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase

    SciTech Connect

    Baldwin, J.; Voegtli, W.C.; Khidekel, N.; Moënne-Loccoz, P.; Krebs, C.; Ley, B.A.; Huynh, B.H.; Loehr, T.M.; Rosenzweig, A.C.; Bollinger, Jr., J.M.

    2010-03-05

    The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of the brown

  12. Structure-Based Design, Synthesis, and Evaluation of 2'-(2-Hydroxyethyl)-2'-deoxyadenosine and the 5'-Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

    SciTech Connect

    Sun, D.; Xu, H.; Wijerathna, S.R.; Dealwis, C.; Lee, R.E.

    2010-08-27

    Analysis of the recently solved X-ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2'-hydroxy group of the nucleoside ribose. In this study 2'-(2-hydroxyethyl)-2'-deoxyadenosine 1 and the 5'-diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2, and the co-crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized.

  13. An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

    PubMed Central

    Conner, J; Cooper, J; Furlong, J; Clements, J B

    1992-01-01

    We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication. Images PMID:1331536

  14. Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

    PubMed Central

    Sun, Y; Conner, J

    2000-01-01

    We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction. PMID:10727407

  15. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

    PubMed Central

    Thelander, L; Berg, P

    1986-01-01

    Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced. Images PMID:3025593

  16. Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

    PubMed Central

    Amara, F M; Chen, F Y; Wright, J A

    1995-01-01

    Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects. Images PMID:7784197

  17. The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site

    PubMed Central

    Aurelius, Oskar; Johansson, Renzo; Bågenholm, Viktoria; Lundin, Daniel; Tholander, Fredrik; Balhuizen, Alexander; Beck, Tobias; Sahlin, Margareta; Sjöberg, Britt-Marie; Mulliez, Etienne; Logan, Derek T.

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga

  18. Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

    PubMed

    Nick, Thomas U; Lee, Wankyu; Kossmann, Simone; Neese, Frank; Stubbe, JoAnne; Bennati, Marina

    2015-01-14

    Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed. PMID:25516424

  19. Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

    PubMed

    Hsieh, Y-Y; Chou, C-J; Lo, H-L; Yang, P-M

    2016-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. PMID:27551518

  20. Expression of Ribonucleotide Reductase Subunit-2 and Thymidylate Synthase Correlates with Poor Prognosis in Patients with Resected Stages I–III Non-Small Cell Lung Cancer

    PubMed Central

    Grossi, Francesco; Dal Bello, Maria Giovanna; Salvi, Sandra; Puzone, Roberto; Pfeffer, Ulrich; Fontana, Vincenzo; Alama, Angela; Rijavec, Erika; Barletta, Giulia; Genova, Carlo; Sini, Claudio; Ratto, Giovanni Battista; Taviani, Mario; Truini, Mauro; Merlo, Domenico Franco

    2015-01-01

    Biomarkers can help to identify patients with early-stages or locally advanced non-small cell lung cancer (NSCLC) who have high risk of relapse and poor prognosis. To correlate the expression of seven biomarkers involved in DNA synthesis and repair and in cell division with clinical outcome, we consecutively collected 82 tumour tissues from radically resected NSCLC patients. The following biomarkers were investigated using IHC and qRT-PCR: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2), subunit p53R2, thymidylate synthase (TS), and class III beta-tubulin (TUBB3). Gene expression levels were also validated in an available NSCLC microarray dataset. Multivariate analysis identified the protein overexpression of RRM2 and TS as independent prognostic factors of shorter overall survival (OS). Kaplan-Meier analysis showed a trend in shorter OS for patients with RRM2, TS, and ERCC1, BRCA1 overexpressed tumours. For all of the biomarkers except TUBB3, the OS trends relative to the gene expression levels were in agreement with those relative to the protein expression levels. The NSCLC microarray dataset showed RRM2 and TS as biomarkers significantly associated with OS. This study suggests that high expression levels of RRM2 and TS might be negative prognostic factors for resected NSCLC patients. PMID:26663950

  1. Function of the Pseudomonas aeruginosa NrdR Transcription Factor: Global Transcriptomic Analysis and Its Role on Ribonucleotide Reductase Gene Expression

    PubMed Central

    Crespo, Anna; Pedraz, Lucas; Torrents, Eduard

    2015-01-01

    Ribonucleotide reductases (RNRs) are a family of sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides (dNTPs), the building blocks for DNA synthesis and repair. Although any living cell must contain one RNR activity to continue living, bacteria have the capacity to encode different RNR classes in the same genome, allowing them to adapt to different environments and growing conditions. Pseudomonas aeruginosa is well known for its adaptability and surprisingly encodes all three known RNR classes (Ia, II and III). There must be a complex transcriptional regulation network behind this RNR activity, dictating which RNR class will be expressed according to specific growing conditions. In this work, we aim to uncover the role of the transcriptional regulator NrdR in P. aeruginosa. We demonstrate that NrdR regulates all three RNR classes, being involved in differential control depending on whether the growth conditions are aerobic or anaerobic. Moreover, we also identify for the first time that NrdR is not only involved in controlling RNR expression but also regulates topoisomerase I (topA) transcription. Finally, to obtain the entire picture of NrdR regulon, we performed a global transcriptomic analysis comparing the transcription profile of wild-type and nrdR mutant strains. The results provide many new data about the regulatory network that controls P. aeruginosa RNR transcription, bringing us a step closer to the understanding of this complex system. PMID:25909779

  2. Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

    PubMed Central

    Hsieh, Y-Y; Chou, C-J; Lo, H-L; Yang, P-M

    2016-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. PMID:27551518

  3. Pseudomonas aeruginosa Exhibits Deficient Biofilm Formation in the Absence of Class II and III Ribonucleotide Reductases Due to Hindered Anaerobic Growth

    PubMed Central

    Crespo, Anna; Pedraz, Lucas; Astola, Josep; Torrents, Eduard

    2016-01-01

    Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this

  4. HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus

    PubMed Central

    Tomter, Ane B.; Zoppellaro, Giorgio; Schmitzberger, Florian; Andersen, Niels H.; Barra, Anne-Laure; Engman, Henrik; Nordlund, Pär; Andersson, K. Kristoffer

    2011-01-01

    Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm−1) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe2+ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class. PMID:21980375

  5. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: adenosylcobalamin destruction and formation of a nucleotide-based radical.

    PubMed

    Lohman, Gregory J S; Gerfen, Gary J; Stubbe, Joanne

    2010-02-23

    Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in <2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C(419) (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u ]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F(2)CTP and the second with AdoCbl destruction. To determine the fate of [1'-(3)H]F(2)CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH(4) (NaB(2)H(4)) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway. PMID:20088568

  6. Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class Ia Binuclear Non-Heme Iron Enzyme

    PubMed Central

    Kwak, Yeonju; Jiang, Wei; Dassama, Laura M.K.; Park, Kiyoung; Bell, Caleb B.; Liu, Lei V.; Wong, Shaun D.; Saito, Makina; Kobayashi, Yasuhiro; Kitao, Shinji; Seto, Makoto; Yoda, Yoshitaka; Alp, E. Ercan; Zhao, Jiyong; Bollinger, J Martin; Krebs, Carsten; Solomon, Edward I.

    2013-01-01

    The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe hetero-binuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable MnIVFeIII cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the MnIVFeIII cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs) / circular dichroism (CD) / magnetic CD (MCD) / variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the FeIII, whereas MCD reflects the spin-allowed transitions mostly on the MnIV. We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with MnIV at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d, and oxo and OH− to metal charge transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the MnIVFeIII cofactor as having a µ-oxo, µ-hydroxo core and a terminal hydroxo ligand on the MnIV. From DFT calculations, the MnIV at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH− terminal ligand on this MnIV provides a high proton affinity that could gate radical translocation to the α (R1) subunit. PMID:24131208

  7. Density Functional Theory Analysis of Structure, Energetics, and Spectroscopy for the Mn-Fe Active Site of Chlamydia trachomatis Ribonucleotide Reductase in Four Oxidation States

    PubMed Central

    Han, Wen-Ge; Giammona, Debra Ann; Bashford, Donald; Noodleman, Louis

    2010-01-01

    Models for the Mn-Fe active site structure of ribonucleotide reductase (RNR) from pathogenic bacteria Chlamydia trachomatis (Ct) in different oxidation states have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and also with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. The detailed structures for the reduced Mn(II)-Fe(II), the met Mn(III)-Fe(III), the oxidized Mn(IV)-Fe(III) and the superoxidized Mn(IV)-Fe(IV) states are predicted. The calculated properties, including geometries, 57Fe Mössbauer isomer shifts and quadrupole splittings, and 57Fe and 55Mn ENDOR hyperfine coupling constants, are compared with the available experimental data. The Mössbauer and energetic calculations show that the (μ-oxo, μ-hydroxo) models better represent the structure of the Mn(IV)-Fe(III) state than the di-μ-oxo models. The predicted Mn(IV)-Fe(III) distances (2.95 and 2.98 Å) in the (μ-oxo, μ-hydroxo) models are in agreement with the EXAFS experimental value of 2.92 Å (Younker, et al. J. Am. Chem. Soc. 2008, 130, 15022-15027). The effect of the protein and solvent environment on the assignment of the Mn metal position is examined by comparing the relative energies of alternative mono-Mn(II) active site structures. It is proposed that if the Mn(II)-Fe(II) protein is prepared with prior addition of Mn(II) or with Mn(II) richer than Fe(II), Mn is likely positioned at metal site 2, which is further from Phe127. PMID:20604534

  8. Use of 2,3,5-F3Y-β2 and 3-NH2Y-α2 to study PCET in E. coli Ribonucleotide Reductase

    PubMed Central

    Seyedsayamdost, Mohammad R.; Yee, Cyril S.; Stubbe, JoAnne

    2011-01-01

    E. coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y122•) cofactor that initiates radical transfer to the active site cysteine in α2 (C439), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y122→W48→Y356 in β2 to Y731→Y730→C439 in α2) and reversible proton coupled electron transfer (PCET). Recently 2,3,5-F3Y (F3Y) was site-specifically incorporated in place of Y356 in β2, and 3-NH2Y (NH2Y) in place of Y731 and Y730 in α2. A pH rate profile with F3Y356-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F3Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH2Y731(730)-α2/β2/CDP/ATP resulted in detection of NH2Y radical (NH2Y•) intermediates capable of dNDP formation. In this study, the reaction of F3Y356-β2/α2/CDP/ATP has been examined by stopped flow (SF) absorption and rapid freeze quench EPR spectroscopy and has failed to reveal any radical intermediates. F3Y356-β2/CDP/ATP has also been examined with NH2Y731-α2 (or NH2Y730-α2) by stopped-flow kinetics from pH 6.5–9.2 and revealed rate constants for NH2Y• formation that support a change in rate limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway. PMID:21182280

  9. A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

    PubMed Central

    Ghosh, Shreya; Samaddar, Sourabh; Kirtania, Prithwiraj

    2015-01-01

    ABSTRACT Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc2155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc2155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCE Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves

  10. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    SciTech Connect

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin

    2014-10-02

    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  11. DFT Calculations of Comparative Energetics and ENDOR/Mössbauer Properties for Two Protonation States of the Iron Dimer Cluster of Ribonucleotide Reductase Intermediate X

    PubMed Central

    Han, Wen-Ge; Noodleman, Louis

    2009-01-01

    Two models (I and II) for the active site structure of class-I ribonucleotide reductase (RNR) intermediate X in subunit R2 have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. Only one of the bridging groups between the two iron centers is different between Model-I and Model-II. Model-I contains two μ-oxo bridges, while Model-II has one bridging oxo and one bridging hydroxo. These are large active site models including up to the fourth coordination shell H-bonding residues. Mössbauer and ENDOR hyperfine property calculations show that Model-I is more likely to represent the active site structure of RNR-X. However, energetically our pKa calculations at first highly favored the bridging oxo and hydroxo (in Model-II) structure of the diiron center rather than having the di-oxo bridge (in Model-I). Since the Arg236 and the nearby Lys42, which are very close to the diiron center, are on the protein surface of RNR-R2, it is highly feasible that one or two anion groups in solution would interact with the positively charged side chains of Arg236 and Lys42. The anion group(s) can be a reductant, phosphate, sulfate, nitrate, and other negatively charged groups existing in biological environment or in the buffer of the experiment. Since sulfate ions certainly exist in the buffer of the ENDOR experiment, we have examined the effect of the sulfate (SO42-, surrounded by explicit water molecules) H-bonding to the side chain of Arg236. We find that when sulfate interacts with Arg236, the carboxylate group of Asp237 tends to be protonated, and once Asp237 is protonated, the Fe(III)Fe(IV) center in X favors the di-oxo bridge (Model-I). This would explain that the ENDOR observed RNR-X active site structure is likely to be represented by Model-I rather than Model-II. PMID

  12. Mechanistic studies of semicarbazone triapine targeting human ribonucleotide reductase in vitro and in mammalian cells: tyrosyl radical quenching not involving reactive oxygen species.

    PubMed

    Aye, Yimon; Long, Marcus J C; Stubbe, JoAnne

    2012-10-12

    Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β(2) subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β(2) have been proposed; one involves direct iron chelation from β(2), whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O(2) and reductant by Fe(II)-(3-AP). Inactivation of β(2) can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of (55)Fe and Y· loss from [((55)Fe(III)(2)-Y·)((55)Fe(III)(2))]-β(2) under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β(2) inactivation. Oxyblot(TM) and mass spectrometric analyses of trypsin-digested inhibited β(2), and lack of Y· loss from H(2)O(2) and O(2)(•) treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β(2) inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(Fe(III)(2)-Y·)(Fe(III)(2))]-β(2) in lysates, similar iron-content in β(2) immunoprecipitated from 3-AP-treated and untreated [(55)Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β(2) are most consistent with Y· loss being the predominant mode of inhibition, with β(2) remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor of β(2). PMID:22915594

  13. Aldose reductase mediates retinal microglia activation.

    PubMed

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. PMID:27033597

  14. Design and synthesis of phosphonoacetic acid (PPA) ester and amide bioisosters of ribofuranosylnucleoside diphosphates as potential ribonucleotide reductase inhibitors and evaluation of their enzyme inhibitory, cytostatic and antiviral activity.

    PubMed

    Manfredini, Stefano; Solaroli, Nicola; Angusti, Angela; Nalin, Federico; Durini, Elisa; Vertuani, Silvia; Pricl, Sabrina; Ferrone, Marco; Spadari, Silvio; Focher, Federico; Verri, Annalisa; De Clercq, Erik; Balzarini, Jan

    2003-07-01

    Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonucleotides to deoxyribonucleotides, we have now prepared and evaluated 5'-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 microM for the UDP analogue 5'-deoxy-5'-N-(phosphon-acetyl)uridine sodium salt (amide) to 600 microM for the CDP analogue 5'-O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100-500 microM concentrations, whereas ADP analogue 5'-N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5'-deoxy-5'-N-(phosphon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 microM) against HSV-2 and a modest inhibitory activity (EC50: 110 microM) against HIV-1, respectively. PMID:14582847

  15. In vivo examination of hydroxyurea and the novel ribonucleotide reductase inhibitors trimidox and didox in combination with the reverse transcriptase inhibitor abacavir: suppression of retrovirus-induced immunodeficiency disease.

    PubMed

    Sumpter, L Ryan; Inayat, Mohammed S; Yost, Erin E; Duvall, William; Hagan, Espen; Mayhew, Christopher N; Elford, Howard L; Gallicchio, Vincent S

    2004-06-01

    Inhibition of ribonucleotide reductase (RR) has gained attention as a potential strategy for HIV-1 therapy through the success of hydroxyurea (HU) to potentiate the activity of the nucleoside reverse transcriptase inhibitor (NRTI) didanosine (ddI) in clinical trials. However, the use of HU has been limited by its development of hematopoietic toxicity. In this study, the novel RR inhibitors didox (DX; 3,4-dihydroxybenzohydroxamic acid), and trimidox (TX; 3,4,5-trihydroxybenzamidoxime) were evaluated along with HU for anti-retroviral efficacy in LPBM5-induced retro-viral disease (MAIDS) both as monotherapeutic regimens and in combination with the guanine containing NRTI abacavir (ABC). Anti-retroviral drug efficacy was determined by measuring inhibition of splenomegaly, hypergammaglobulinemia, and splenic levels of proviral DNA. In this study, all RRIs tested showed the ability to improve the efficacy of ABC in the MAIDS model by reducing splenomegaly, hypergammaglobulinemia, and splenic proviral DNA levels. PMID:15130534

  16. Comparison of the expression of human equilibrative nucleotide transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) genes in seven non-Hodgkin lymphoma cell lines.

    PubMed

    Zhao, H B; Zhang, X F; Shi, F; Zhang, M Z; Xue, W L

    2016-01-01

    We investigated the variability in the expression of human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) in non-Hodgkin lymphoma cell lines. hENT1 and RRM1 mRNA expression levels in natural killer (NK) cells and seven non-Hodgkin lymphoma cell lines (YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, and Jurket) were studied using reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) and the results were compared using the Student t-test. mRNA expression of hENT1 was detectable in YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, Jurket, and NK cells, which revealed variability in gene expression. There were significant differences in the mRNA expression values of hENT1 (P = 0.021) and RRM1 (P = 0.002) compared to those in NK cells. mRNA expression of both hENT1 and RRM1 was closely associated with non-Hodgkin lymphoma cell proliferation. Differential expression analysis of hENT1 and RRM1 in non-Hodgkin lymphoma cell lines may provide novel drug leads for precision medicine. PMID:27173327

  17. A PHASE I STUDY OF THE NOVEL RIBONUCLEOTIDE REDUCTASE INHIBITOR 3-AMINOPYRIDINE-2-CARBOXALDEHYDE THIOSEMICARBAZONE (3-AP, TRIAPINE®) IN COMBINATION WITH THE NUCLEOSIDE ANALOG FLUDARABINE FOR PATIENTS WITH REFRACTORY ACUTE LEUKEMIAS AND AGGRESSIVE MYELOPROLIFERATIVE DISORDERS

    PubMed Central

    Karp, Judith E.; Giles, Francis J.; Gojo, Ivana; Morris, Lawrence; Greer, Jacqueline; Johnson, Bonny; Thein, Mya; Sznol, Mario; Low, Jennifer

    2009-01-01

    Triapine® is a potent ribonucleotide reductase (RR) inhibitor that depletes intracellular deoxyribonculeotide pools, especially dATP. We designed a Phase I trial of Triapine followed by the adenosine analog fludarabine in adults with refractory acute leukemias and aggressive myeloproliferative disorders (MPD). Two schedules were examined: A. Triapine 105mg/m2/day over 4 hours followed by fludarabine daily × 5 (24 patients, fludarabine 15–30 mg/m2/dose); B. Triapine 200mg/m2 over 24 hours followed by 5 days of fludarabine 30 mg/m2/day (9 patients). Complete and partial responses (CR,PR) occurred in Schedule A (5/24, 21%), with CR occurring at the 2 highest fludarabine doses (2/12, 17%). In contrast, no CR or PR occurred in Schedule B. Four of the 5 responses occurred in patients with underlying MPD (4/14, 29%). Drug-related toxicities included fever and metabolic acidosis. Triapine 105 mg/m2 followed by fludarabine 30mg/m2 daily × 5 is active in refractory myeloid malignancies and warrants continuing study for patients with aggressive MPD. PMID:17640728

  18. Structural Analysis of the Mn(IV)/Fe(III) Cofactor of Chlamydia trachomatis Ribonucleotide Reductase by Extended X-ray Absorption Fine Structure Spectroscopy and Density Functional Theory Calculations

    PubMed Central

    Younker, Jarod M.; Krest, Courtney M.; Jiang, Wei; Krebs, Carsten; Bollinger, J. Martin; Green, Michael T.

    2009-01-01

    The class Ic ribonucleotide reductase from Chlamydia trachomatis (Ct) uses a stable Mn(IV)/Fe(III) cofactor to initiate nucleotide reduction by a free-radical mechanism. Extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) calculations are used to postulate a structure for this cofactor. Fe and Mn K-edge EXAFS data yield an intermetallic distance of ~2.92 Å. The Mn data also suggest the presence of a short 1.74 Å Mn—O bond. These metrics are compared to the results of DFT calculations on 12 cofactor models derived from the crystal structure of the inactive Fe2(III/III) form of the protein. Models are differentiated by the protonation states of their bridging and terminal OHX ligands as well as the location of the Mn(IV) ion (site 1 or 2). The models that agree best with experimental observation feature a µ-1,3-carboxylate bridge (E120), terminal solvent (H2O/OH) to site 1, one µ-O bridge, and one µ-OH bridge. The site-placement of the metal ions cannot be discerned from the available data. PMID:18937466

  19. Structural Analysis of the Mn(IV)/Fe(III) Cofactor of Chlamydia Trachomatis Ribonucleotide Reductase By Extended X-Ray Absorption Fine Structure Spectroscopy And Density Functional Theory Calculations

    SciTech Connect

    Younker, J.M.; Krest, C.M.; Jiang, W.; Krebs, C.; Bollinger, J.M.Jr.; Green, M.T.

    2009-05-28

    The class Ic ribonucleotide reductase from Chlamydia trachomatis (C{bar A}) uses a stable Mn(lV)/ Fe(lll) cofactor to initiate nucleotide reduction by a free-radical mechanism. Extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) calculations are used to postulate a structure for this cofactor. Fe and Mn K-edge EXAFS data yield an intermetallic distance of -2.92 {angstrom}. The Mn data also suggest the presence of a short 1.74 {angstrom} Mn-O bond. These metrics are compared to the results of DFT calculations on 12 cofactor models derived from the crystal structure of the inactive Fe2(lll/ III) form of the protein. Models are differentiated by the protonation states of their bridging and terminal OH{sub x} ligands as well as the location of the Mn(lV) ion (site 1 or 2). The models that agree best with experimental observation feature a{mu}-1, 3-carboxylate bridge (E120), terminal solvent (H{sub 2}O/OH) to site 1, one {mu}-O bridge, and one {mu}-OH bridge. The site-placement of the metal ions cannot be discerned from the available data.

  20. Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate

    PubMed Central

    Cotruvo, Joseph A.; Stich, Troy A.; Britt, R. David; Stubbe, JoAnne

    2013-01-01

    Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y• cofactor in their NrdF subunit. MnII2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•− (40-48 s−1, 0.6 mM O2), the O2•− channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s−1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08-0.15 s−1). Controlled production of O2•− by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII2 cluster with O2 and satisfies the requirement for an “extra” reducing equivalent in Y• generation. PMID:23402532

  1. Spectroscopic and Electronic Structure Studies of Intermediate X in Ribonucleotide Reductase R2 and Two Variants: A Description of the FeIV-Oxo Bond in the FeIII-O-FeIV Dimer

    PubMed Central

    Mití, Nataša; Clay, Michael D.; Saleh, Lana; Solomon, Edward I.

    2008-01-01

    Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally-derived model complexes are presented, compared and evaluated to determine the electronic and geometric structure of the FeIII-FeIV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the FeIII/FeIV active site, which displayed three FeIV d-d transitions between 16 700 – 22 600 cm-1, two FeIV d-d spin-flip transitions between 23 500 – 24 300 cm-1 and five oxo to FeIV and FeIII charge transfer (CT) transitions between 25 000 – 32 000 cm-1. The FeIV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-π manifold. Furthermore, the FeIV d-π splittings in the WT are too large to correlate with a bis-μ-oxo structure. The assignment of the FeIV d-d transitions in WT intermediate X best correlates with a bridged μ-oxo/μ-hydroxo [FeIII(μ-O)(μ-OH)FeIV] structure. The μ-oxo/μ-hydroxo core structure provides an important σ/π superexchange pathway, which is not present in the bis-μ-oxo structure, to promote facile electron transfer from Y122 to the remote FeIV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis. PMID:17602477

  2. Site-specific insertion of 3-aminotyrosine into subunit alpha2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation.

    PubMed

    Seyedsayamdost, Mohammad R; Xie, Jianming; Chan, Clement T Y; Schultz, Peter G; Stubbe, JoAnne

    2007-12-01

    E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover. PMID:17990884

  3. The Origin and Evolution of Ribonucleotide Reduction

    PubMed Central

    Lundin, Daniel; Berggren, Gustav; Logan, Derek T.; Sjöberg, Britt-Marie

    2015-01-01

    Ribonucleotide reduction is the only pathway for de novo synthesis of deoxyribonucleotides in extant organisms. This chemically demanding reaction, which proceeds via a carbon-centered free radical, is catalyzed by ribonucleotide reductase (RNR). The mechanism has been deemed unlikely to be catalyzed by a ribozyme, creating an enigma regarding how the building blocks for DNA were synthesized at the transition from RNA- to DNA-encoded genomes. While it is entirely possible that a different pathway was later replaced with the modern mechanism, here we explore the evolutionary and biochemical limits for an origin of the mechanism in the RNA + protein world and suggest a model for a prototypical ribonucleotide reductase (protoRNR). From the protoRNR evolved the ancestor to modern RNRs, the urRNR, which diversified into the modern three classes. Since the initial radical generation differs between the three modern classes, it is difficult to establish how it was generated in the urRNR. Here we suggest a model that is similar to the B12-dependent mechanism in modern class II RNRs. PMID:25734234

  4. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  5. Mössbauer Properties of the Diferric Cluster and the Differential Iron(II)-Binding Affinity of the Iron Sites in Protein R2 of Class Ia Escherichia coli Ribonucleotide Reductase: A DFT/Electrostatics Study

    PubMed Central

    Han, Wen-Ge; Sandala, Gregory M.; Giammona, Debra Ann; Bashford, Donald; Noodleman, Louis

    2013-01-01

    The R2 subunit of class-Ia ribonucleotide reductase (RNR) from Escherichia coli (E. coli) contains a diiron active site. Starting from the apo-protein and Fe(II) in solution at low Fe(II)/apoR2 ratios, mononuclear Fe(II) binding is observed indicating possible different Fe(II) binding affinities for the two alternative sites. Further, based on their Mössbauer spectroscopy and two-iron-isotope reaction experiments, Bollinger et al. (J. Am. Chem. Soc., 1997, 119, 5976–5977) proposed that the site Fe1, which bonds to Asp84, should be associated with the higher observed 57Fe Mössbauer quadrupole splitting (2.41 mm s−1) and lower isomer shift (0.45 mm s−1) in the Fe(III)Fe(III) state, site Fe2, which is further from Tyr122, should have a greater affinity for Fe(II) binding than site Fe1, and Fe(IV) in the intermediate X state should reside at site Fe2. In this paper, using density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) methodologies, we have demonstrated that the observed large quadrupole splitting for the diferric state R2 does come from site Fe1(III) and it is mainly caused by the binding position of the carboxylate group of Asp84 sidechain. Further, a series of active site clusters with mononuclear Fe(II) binding at either site Fe1 or Fe2 have been studied, which show that with single dielectric medium outside the active site quantum region, there is no energetic preference for Fe(II) binding at one site over another. However, when including the explicit extended protein environment in the PB-SCRF model, the reaction field favors the Fe(II) binding at site Fe2 rather than at site Fe1 by ~9 kcal mol−1. Therefore our calculations support the proposal of the previous Mössbauer spectroscopy and two-iron-isotope reaction experiments by Bollinger et al. PMID:21837345

  6. DNA Polymerase β Ribonucleotide Discrimination

    PubMed Central

    Cavanaugh, Nisha A.; Beard, William A.; Wilson, Samuel H.

    2010-01-01

    DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion. PMID:20519499

  7. The Balancing Act of Ribonucleotides in DNA.

    PubMed

    Cerritelli, Susana M; Crouch, Robert J

    2016-05-01

    The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the ribonucleotide excision repair (RER) pathway, a process similar to Okazaki fragment (OF) processing after incision by Ribonuclease H2 (RNase H2). All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when, and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA. We also review the data that indicate that removal of ribonucleotides has an important role in maintaining genome stability. PMID:26996833

  8. Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells

    SciTech Connect

    Kleinberger, T.; Etkin, S.; Lavi, S.

    1986-06-01

    We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

  9. Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2

    PubMed Central

    Kim, Joo Yong; Kwon, Ye Jin; Kim, Sung-Il; Kim, Do Youn; Song, Jong Tae; Seo, Hak Soo

    2016-01-01

    Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation. PMID:26834755

  10. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  11. Processing ribonucleotides incorporated during eukaryotic DNA replication.

    PubMed

    Williams, Jessica S; Lujan, Scott A; Kunkel, Thomas A

    2016-06-01

    The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease. PMID:27093943

  12. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells

    PubMed Central

    Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-01-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  13. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells.

    PubMed

    Zou, Peng; Xia, Yiqun; Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-04-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer effïcacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  14. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  15. The Nitric Oxide Production in the Moss Physcomitrella patens Is Mediated by Nitrate Reductase

    PubMed Central

    Medina-Andrés, Rigoberto; Solano-Peralta, Alejandro; Saucedo-Vázquez, Juan Pablo; Napsucialy-Mendivil, Selene; Pimentel-Cabrera, Jaime Arturo; Sosa-Torres, Martha Elena; Dubrovsky, Joseph G.; Lira-Ruan, Verónica

    2015-01-01

    During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae. PMID:25742644

  16. The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

    PubMed

    Medina-Andrés, Rigoberto; Solano-Peralta, Alejandro; Saucedo-Vázquez, Juan Pablo; Napsucialy-Mendivil, Selene; Pimentel-Cabrera, Jaime Arturo; Sosa-Torres, Martha Elena; Dubrovsky, Joseph G; Lira-Ruan, Verónica

    2015-01-01

    During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae. PMID:25742644

  17. Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney

    PubMed Central

    Hu, Zhizhi; Pei, Guangchang; Wang, Pengge; Yang, Juan; Zhu, Fengmin; Guo, Yujiao; Wang, Meng; Yao, Ying; Zeng, Rui; Liao, Wenhui; Xu, Gang

    2015-01-01

    Biliverdin reductase A is an enzyme, with serine/threonine/tyrosine kinase activation, converting biliverdin (BV) to bilirubin (BR) in heme degradation pathway. It has been reported to have anti-inflammatory and antioxidant effect in monocytes and human glioblastoma. However, the function of BVRA in polarized macrophage was unknown. This study aimed to investigate the effect of BVRA on macrophage activation and polarization in injured renal microenvironment. Classically activated macrophages (M1macrophages) and alternative activation of macrophages (M2 macrophages) polarization of murine bone marrow derived macrophage was induced by GM-CSF and M-CSF. M1 polarization was associated with a significant down-regulation of BVRA and Interleukin-10 (IL-10), and increased secretion of TNF-α. We also found IL-10 expression was increased in BVRA over-expressed macrophages, while it decreased in BVRA knockdown macrophages. In contrast, BVRA over-expressed or knockdown macrophages had no effect on TNF-α expression level, indicating BVRA mediated IL-10 expression in macrophages. Furthermore, we observed in macrophages infected with recombinant adenoviruses BVRA gene, which BVRA over-expressed enhanced both INOS and ARG-1 mRNA expression, resulting in a specific macrophage phenotype. Through in vivo study, we found BVRA positive macrophages largely existed in mice renal ischemia perfusion injury. With the treatment of the regular cytokines GM-CSF, M-CSF or LPS, excreted in the injured renal microenvironment, IL-10 secretion was significantly increased in BVRA over-expressed macrophages. In conclusion, the BVRA positive macrophage is a source of anti-inflammatory cytokine IL-10 in injured kidney, which may provide a potential target for treatment of kidney disease. PMID:26393580

  18. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  19. Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

    PubMed Central

    Pugin, Benoit; Cornejo, Fabián A.; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M.; Vargas-Pérez, Joaquín I.; Arenas, Felipe A.

    2014-01-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. PMID:25193000

  20. Glutathione reductase-mediated synthesis of tellurium-containing nanostructures exhibiting antibacterial properties.

    PubMed

    Pugin, Benoit; Cornejo, Fabián A; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M; Vargas-Pérez, Joaquín I; Arenas, Felipe A; Vásquez, Claudio C

    2014-11-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. PMID:25193000

  1. Nitrate reductase-mediated NO production enhances Cd accumulation in Panax notoginseng roots by affecting root cell wall properties.

    PubMed

    Kan, Qi; Wu, Wenwei; Yu, Wenqian; Zhang, Jiarong; Xu, Jin; Rengel, Zed; Chen, Limei; Cui, Xiuming; Chen, Qi

    2016-04-01

    Panax notoginseng (Burk) F. H. Chen is a traditional medicinal herb in China. However, the high capacity of its roots to accumulate cadmium (Cd) poses a potential risk to human health. Although there is some evidence for the involvement of nitric oxide (NO) in mediating Cd toxicity, the origin of Cd-induced NO and its function in plant responses to Cd remain unknown. In this study, we examined NO synthesis and its role in Cd accumulation in P. notoginseng roots. Cd-induced NO production was significantly decreased by application of the nitrate reductase inhibitor tungstate but not the nitric oxide synthase inhibitor L-NAME (N(G)-methyl-l-arginine acetate), indicating that nitrate reductase is the major contributor to Cd-induced NO production in P. notoginseng roots. Under conditions of Cd stress, sodium nitroprusside (SNP, an NO donor) increased Cd accumulation in root cell walls but decreased Cd translocation to the shoot. In contrast, the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and tungstate both significantly decreased NO-increased Cd retention in root cell walls. The amounts of hemicellulose 1 and pectin, together with pectin methylesterase activity, were increased with the addition of SNP but were decreased by cPTIO and tungstate. Furthermore, increases or decreases in hemicellulose 1 and pectin contents as well as pectin methylesterase activity fit well with the increased or decreased retention of Cd in the cell walls of P. notoginseng roots. The results suggest that nitrate reductase-mediated NO production enhances Cd retention in P. notoginseng roots by modulating the properties of the cell wall. PMID:26956919

  2. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

    PubMed Central

    Ding, Bo; Gibbs, Peter E. M.; Brookes, Paul S.; Maines, Mahin D.

    2011-01-01

    HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca2-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC281 and KYCCSRK296, respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival; a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

  3. Error-free and mutagenic processing of topoisomerase 1-provoked damage at genomic ribonucleotides

    PubMed Central

    Sparks, Justin L; Burgers, Peter M

    2015-01-01

    Genomic ribonucleotides incorporated during DNA replication are commonly repaired by RNase H2-dependent ribonucleotide excision repair (RER). When RNase H2 is compromised, such as in Aicardi-Goutières patients, genomic ribonucleotides either persist or are processed by DNA topoisomerase 1 (Top1) by either error-free or mutagenic repair. Here, we present a biochemical analysis of these pathways. Top1 cleavage at genomic ribonucleotides can produce ribonucleoside-2′,3′-cyclic phosphate-terminated nicks. Remarkably, this nick is rapidly reverted by Top1, thereby providing another opportunity for repair by RER. However, the 2′,3′-cyclic phosphate-terminated nick is also processed by Top1 incision, generally 2 nucleotides upstream of the nick, which produces a covalent Top1–DNA complex with a 2-nucleotide gap. We show that these covalent complexes can be processed by proteolysis, followed by removal of the phospho-peptide by Tdp1 and the 3′-phosphate by Tpp1 to mediate error-free repair. However, when the 2-nucleotide gap is associated with a dinucleotide repeat sequence, sequence slippage re-alignment followed by Top1-mediated religation can occur which results in 2-nucleotide deletion. The efficiency of deletion formation shows strong sequence-context dependence. PMID:25777529

  4. The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase*

    PubMed Central

    Zhang, Min; Zhang, Feilong; Fang, Ying; Chen, Xuemin; Chen, Yuhong; Zhang, Wenxia; Dai, Huai-En; Lin, Rongcheng; Liu, Lin

    2015-01-01

    The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLUTPR) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLUTPR at 2.4-Å resolution. Three non-canonical TPR motifs of each FLUTPR form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLUTPR-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein. PMID:26037924

  5. GENERAL CONTROL NONREPRESSED PROTEIN5-Mediated Histone Acetylation of FERRIC REDUCTASE DEFECTIVE3 Contributes to Iron Homeostasis in Arabidopsis1

    PubMed Central

    Xing, Jiewen; Wang, Tianya; Liu, Zhenshan; Xu, Jianqin; Yao, Yingyin; Hu, Zhaorong; Peng, Huiru; Xin, Mingming; Yu, Futong; Zhou, Daoxiu; Ni, Zhongfu

    2015-01-01

    Iron homeostasis is essential for plant growth and development. Here, we report that a mutation in GENERAL CONTROL NONREPRESSED PROTEIN5 (GCN5) impaired iron translocation from the root to the shoot in Arabidopsis (Arabidopsis thaliana). Illumina high-throughput sequencing revealed 879 GCN5-regulated candidate genes potentially involved in iron homeostasis. Chromatin immunoprecipitation assays indicated that five genes (At3G08040, At2G01530, At2G39380, At2G47160, and At4G05200) are direct targets of GCN5 in iron homeostasis regulation. Notably, GCN5-mediated acetylation of histone 3 lysine 9 and histone 3 lysine 14 of FERRIC REDUCTASE DEFECTIVE3 (FRD3) determined the dynamic expression of FRD3. Consistent with the function of FRD3 as a citrate efflux protein, the iron retention defect in gcn5 was rescued and fertility was partly restored by overexpressing FRD3. Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate. Collectively, these data suggest that GCN5 plays a critical role in FRD3-mediated iron homeostasis. Our results provide novel insight into the chromatin-based regulation of iron homeostasis in Arabidopsis. PMID:26002909

  6. Thioredoxin reductase 1 ablation sensitizes colon cancer cells to methylseleninate-mediated cytotoxicity

    SciTech Connect

    Honeggar, Matthew; Beck, Robert; Moos, Philip J.

    2009-12-15

    The relationship between selenium and cancer is complex because individuals with low serum selenium levels benefit from selenium supplementation, but those with high serum selenium levels are at increased risk for other diseases. This suggests that the use of selenocompounds might be limited to particular circumstances, such as adjuvant therapy. A contributor to this dichotomy may be the activity of certain selenium containing enzymes like the cytosolic thioredoxin reductase (TR1). We evaluated the cellular response to select selenocompounds that have anticancer activity when TR1 was attenuated by siRNA in RKO colon cancer cells. Methylseleninic acid (MSA), which is a substrate for TR1, enhanced cytotoxicity to colon cancer cells when TR1 was attenuated. MSA induced stress in the endoplasmic reticulum, as measured by GRP78 protein levels. However, this pathway did not appear to account for the change in cytotoxicity when TR1 was attenuated. Instead, knockdown of the cytosolic TR plus incubation with MSA increased autophagy, as measured by LC3B cleavage, and apoptosis, as measured by Annexin V and mitochondrial dysfunction. Therefore, the use of selenocompounds with anticancer activity, like MSA, might be utilized most effectively with agents that targets TR1 in chemotherapeutic applications.

  7. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene

    SciTech Connect

    Ricciardone, M.D.

    1986-01-01

    Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.

  8. Aldose Reductase-Mediated Phosphorylation of p53 Leads to Mitochondrial Dysfunction, and Damage in Diabetic Platelets

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Jin, Yu; Liu, Renjing; Lee, Seung Hee; Du, Jing; Atteya, Gourg; Gleim, Scott; Spollett, Geralyn; Martin, Kathleen; Hwa, John

    2014-01-01

    Background Platelet abnormalities are well-recognized complications of diabetes mellitus (DM). Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in DM. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in DM platelets is unknown. Methods and Results Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase (AR) activation, and subsequent reactive oxygen species (ROS) production, leads to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage and rupture by sequestration of the anti-apoptotic protein Bcl-xL. In a glucose dose dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, AR activation, ROS production and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are two distinct states, dependent upon the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model. Conclusions AR contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for DM thrombotic complications. PMID:24474649

  9. A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas[W

    PubMed Central

    de Montaigu, Amaury; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2010-01-01

    Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants. PMID:20442374

  10. Farnesol is not the nonsterol regulator mediating degradation of HMG-CoA reductase in rat liver.

    PubMed

    Keller, R K; Zhao, Z; Chambers, C; Ness, G C

    1996-04-15

    A recent report, in which cultured tumor cells were used, identified farnesol as the nonsterol mevalonate-derived metabolite required for the accelerated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (C. C. Correll, L. Ng, and P. A. Edwards, 1994, J. Biol. Chem. 269, 17390-17393). We examined this proposed linkage in animals by measuring hepatic farnesol levels and rates of HMG-CoA reductase degradation under conditions previously shown to alter the stability of the reductase. In normal rats, the hepatic farnesol level, quantified by high-pressure liquid chromatography, was 0.10 +/- 0.08 microgram/g and the half-life of HMG-CoA reductase was 2.5 h. Administration of mevalonolactone at 1 g/kg body wt to provide all nonsterol metabolites in addition to cholesterol increased farnesol levels 6-fold without significantly affecting the half-life of the reductase. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, raised hepatic farnesol levels 10-fold and decreased the half-life of HMG-CoA reductase to 0.25 h. However, feeding lovastatin to rats did not lower hepatic farnesol levels despite a marked stabilization of HMB-CoA reductase protein. Moreover, intubation of rats with 500 mg/kg body wt of farnesol failed to decrease the half-life of HMG-CoA reductase protein, alter the levels of enzyme activity, or change of the levels of immunoreactive protein despite an increase of 1000-fold in hepatic farnesol levels. These observations indicate that farnesol per se does not induce accelerated degradation of HMG-CoA reductase in rat liver. PMID:8645011

  11. Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease

    PubMed Central

    SAMUELS, IVY S.; LEE, CHIEH-ALLEN; PETRASH, J. MARK; PEACHEY, NEAL S.; KERN, TIMOTHY S.

    2013-01-01

    Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR−/− mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR−/− diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR−/− mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR−/− animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR−/− diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs. PMID:23101909

  12. Human Vitamin K 2,3-Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Mediates Vitamin K-dependent Intracellular Antioxidant Function*

    PubMed Central

    Westhofen, Philipp; Watzka, Matthias; Marinova, Milka; Hass, Moritz; Kirfel, Gregor; Müller, Jens; Bevans, Carville G.; Müller, Clemens R.; Oldenburg, Johannes

    2011-01-01

    Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: Km (μm) = 4.15 (vitamin K1 epoxide) and 11.24 (vitamin K2 epoxide); Vmax (nmol·mg−1·hr−1) = 2.57 (vitamin K1 epoxide) and 13.46 (vitamin K2 epoxide). Oxidative stress induced by H2O2 applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K1 and K2 but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K1. Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival. PMID:21367861

  13. Anthracycline resistance mediated by reductive metabolism in cancer cells: The role of aldo-keto reductase 1C3

    SciTech Connect

    Hofman, Jakub; Malcekova, Beata; Skarka, Adam; Novotna, Eva; Wsol, Vladimir

    2014-08-01

    Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2′-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment. - Highlights: • Metabolism of anthracyclines by AKR1C3 was studied at enzyme and cellular levels. • Anthracycline resistance mediated by AKR1C3 was demonstrated in cancer cells. • Induction of AKR1C3

  14. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution.

    PubMed

    Cha, Joon-Yung; Kim, Mi R; Jung, In J; Kang, Sun B; Park, Hee J; Kim, Min G; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OX(C85S) plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OX(C85S) plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  15. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution

    PubMed Central

    Cha, Joon-Yung; Kim, Mi R.; Jung, In J.; Kang, Sun B.; Park, Hee J.; Kim, Min G.; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OXC85S plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OXC85S plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  16. Di-(2-ethylhexyl) phthalate inhibits testosterone level through disturbed hypothalamic-pituitary-testis axis and ERK-mediated 5α-Reductase 2.

    PubMed

    Ha, Mei; Guan, Xie; Wei, Li; Li, Peng; Yang, Min; Liu, Changjiang

    2016-09-01

    Di-(2-ethylhexyl) phthalate (DEHP) has reproductive toxicity and can affect male reproductive development. In order to clarify adverse effects of DEHP on testicular physiology and testosterone production, Sprague-Dawley (SD) rats were dosed daily with DEHP by gavage for 30days; TM3 cells (mouse Leydig cell line) were treated with DEHP for 24h after pretreatment with vitamin C or U0126. Results indicated that the hypothalamic-pituitary-testis (HPT) axis was disturbed and serum testosterone, LH and FSH levels were decreased following DEHP exposure. Histomorphological changes of rat testes were also observed, such as deformed seminiferous tubules, aggregated chromatin, multiple vacuoles, swollen mitochondria, apoptotic germ cells and Sertoli cells, as well as increased Leydig cell numbers. Moreover, DEHP caused oxidative stress in vivo and in vitro and then induced the ERK pathway, which was required to mediate 5α-Reductase 2 and scavenger receptor class B-1 (SRB1) levels. However, levels of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), P450 17α-hydroxylase/17.20 lyase (P450c17), and P450 side-chain cleavage enzyme (P450scc) were not significantly altered after DEHP exposure. Taken together, DEHP-disturbed HPT axis and induced 5α-Reductase 2 contribute to the reduction of serum testosterone level. The activated ERK pathway is required to modulate expressions of 5α-Reductase 2 and SRB1. PMID:27155079

  17. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  18. A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana

    PubMed Central

    Desikan, Radhika; Griffiths, Rachael; Hancock, John; Neill, Steven

    2002-01-01

    The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in Arabidopsis guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type Arabidopsis generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant, nia1, nia2 that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI) abi1–1 and abi2–1 mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in Arabidopsis guard cells. PMID:12446847

  19. Uveal Melanoma Cell Growth Is Inhibited by Aminoimidazole Carboxamide Ribonucleotide (AICAR) Partially Through Activation of AMP-Dependent Kinase

    PubMed Central

    Al-Moujahed, Ahmad; Nicolaou, Fotini; Brodowska, Katarzyna; Papakostas, Thanos D.; Marmalidou, Anna; Ksander, Bruce R.; Miller, Joan W.; Gragoudas, Evangelos; Vavvas, Demetrios G.

    2014-01-01

    Purpose. To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Methods. Four different cell lines were treated with AICAR (1–4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Results. Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5′-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5′-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Conclusions. Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1. PMID:24781943

  20. Antihyperlipidemic Activity of Aloe succotrina in Rats: Possibly Mediated by Inhibition of HMG-CoA Reductase

    PubMed Central

    Lamba, Deepak; Kumar, Ramesh; Nath, Pashupati; Gauttam, Satyaprakash

    2014-01-01

    The present study was designed to investigate antihyperlipidemic activity of dried pulp of Aloe succotrina leaves in Wistar albino rats. Hyperlipidemia was induced in rats by feeding them high fat diet (HFD) or D-fructose (25% w/v) for 4 successive weeks. From 15th to 28th day, dried pulp (100 and 200 mg/kg, p.o) and atorvastatin (10 mg/kg, p.o.) per se were administered 2 h prior to feeding rats with HFD or fructose. Aloe succotrina did not significantly decrease the body weight of rats. The dried pulp and atorvastatin per se significantly decreased relative liver weight but did not significantly affect relative heart weight. HFD or fructose significantly increased serum total cholesterol, triglycerides, LDL-c, and VLDL, and decreased HDL-c; significantly increased liver MDA and decreased GSH levels. The dried pulp (200 mg/kg p.o.) significantly reversed high fat diet-induced and fructose-induced hyperlipidemia and atherogenic index. Aloe succotrina significantly decreased HMG Co-A reductase activity. Antihyperlipidemic effect of the dried pulp was comparable to atorvastatin. Thus, Aloe succotrina produced significant antihyperlipidemic activity in both HFD and fructose-induced hyperlipidemic rats, possibly through normalization of serum lipid profile, HMG-CoA reductase inhibitory activity, and amelioration of oxidative stress in liver. PMID:24693447

  1. Inhibition of geranylgeranylation mediates the effects of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors on microglia.

    PubMed

    Bi, Xiaoning; Baudry, Michel; Liu, Jihua; Yao, Yueqin; Fu, Lawrence; Brucher, Fernando; Lynch, Gary

    2004-11-12

    Inflammatory responses involving microglia, the resident macrophages of the brain, are thought to contribute importantly to the progression of Alzheimer's disease (AD) and possibly other neurodegenerative disorders. The present study tested whether the mevalonate-isoprenoid biosynthesis pathway, which affects inflammation in many types of tissues, tonically regulates microglial activation. This question takes on added significance given the potential use of statins, drugs that block the rate-limiting step (3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)) in mevalonate and cholesterol synthesis, in AD treatment. Both mevastatin and simvastatin caused a concentration- and time-dependent activation of microglia in cultured rat hippocampal slices. This response consisted of a transformation of the cells from a typical resting configuration to an amoeboid, macrophage-like morphology, increased expression of a macrophage antigen, and up-regulation of the cytokine tumor necrosis factor-alpha. Evidence for proliferation was also obtained. Statin-induced microglial changes were blocked by mevalonate but not by cholesterol, indicating that they were probably due to suppression of isoprenoid synthesis. In accord with this, the statin effects were absent in slices co-incubated with geranylgeranyl pyrophosphate, a mevalonate product that provides for the prenylation of Rho GTPases. Finally, PD98089, a compound that blocks activation of extracellularly regulated kinases1/2, suppressed statin-induced up-regulation of tumor necrosis factor-alpha but had little effect on microglial transformation. These results suggest that 1) the mevalonate-isoprenoid pathway is involved in regulating microglial morphology and in controlling expression of certain cytokines and 2) statins have the potential for enhancing a component of AD with uncertain relationships to other features of the disease. PMID:15364922

  2. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120

    PubMed Central

    Sánchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, Carla V.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments. PMID:27588019

  3. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; Florencio, Francisco J

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments. PMID:27588019

  4. Stretch Moduli of Ribonucleotide Embedded Short DNAs

    NASA Astrophysics Data System (ADS)

    Chiu, Hsiang-Chih; Koh, Kyung Duk; Riedo, Elisa; Storici, Francesca

    2013-03-01

    Understanding the mechanical properties of DNA is essential to comprehending the dynamics of many cellular functions. DNA deformations are involved in many mechanisms when genetic information needs to be stored and used. In addition, recent studies have found that Ribonucleotides (rNMPs) are among the most common non-standard nucleotides present in DNA. The presences of rNMPs in DNA might cause mutation, fragility or genotoxicity of chromosome but how they influence the structure and mechanical properties of DNA remains unclear. By means of Atomic Force Microscopy (AFM) based single molecule spectroscopy, we measure the stretch moduli of double stranded DNAs (dsDNA) with 30 base pairs and 5 equally embedded rNMPs. The dsDNAs are anchored on gold substrate via thiol chemistry, while the AFM tip is used to pick up and stretch the dsDNA from its free end through biotin-streptavidin bonding. Our preliminary results indicate that the inclusion of rNMPs in dsDNA might significantly change its stretch modulus, which might be important in some biological processes.

  5. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    SciTech Connect

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  6. Ribonucleotide reduction - horizontal transfer of a required function spans all three domains

    PubMed Central

    2010-01-01

    Background Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes. Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT. Conclusions Horizontal gene transfer has clearly played an

  7. The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Ravid, T; Doolman, R; Avner, R; Harats, D; Roitelman, J

    2000-11-17

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR. PMID:10964918

  8. Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells.

    PubMed

    Rao, Abhi K; Ziegler, Yvonne S; McLeod, Ian X; Yates, John R; Nardulli, Ann M

    2009-12-01

    Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERalpha, Trx, and TrxR interact and ERalpha and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17beta-estradiol, Trx, and TrxR alter hydrogen peroxide (H(2)O(2)) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H(2)O(2) levels and transcription factor activity, aid ERalpha in regulating the expression of estrogen-responsive genes in target cells. PMID:19620238

  9. Effects of various compounds on lipid peroxidation mediated by detergent-solubilized rat liver NADPH-cytochrome C reductase.

    PubMed

    Kamataki, T; Sugita, O; Naminohira, S; Kitagawa, H

    1978-12-01

    A reconstituted lipid peroxidation system containing NADPH-cytochrome c reductase isolated from detergent-solubilized rat liver microsomes was used to determine the effects of several compounds, including drugs, on the lipid peroxidation activity. EDTA and ferrous ion were essential requirements for reconstitution of the activity. The addition of 1,10-phenanthroline to the system containing both EDTA and ferrous ion further enhanced the activity. Pyrocatecol, thymol, p-aminophenol, imipramine, p-chloromercuribenzoate (PCMB) and alpha-tocopherol exhibited strong inhibition, aniline, N-monomethylaniline, aminopyrine, benzphetamine, SKF 525-A and NADP exhibited moderate inhibition, and phenol, benzoic acid, acetanilide and nicotinamide exhibited less or no inhibition at the concentrations lower than 1000 micron M. Metal ions such as Hg+, Hg2+, Co2+, Cu2+, Mn2+ and U6+ inhibited lipid peroxidation strongly. In addition, Cd2+, St2+ and Ca2+ exhibited less potent to moderate inhibition, and Ba2+ and Mg2+ were without effects on the activity. Among sulfhydryl compounds tested, dithiothreitol inhibited lipid peroxidation to a greater extent than did the other three compounds, glutathione, cysteine and mercaptoethanol. PMID:106178

  10. Inhibition of thioredoxin reductase by alantolactone prompts oxidative stress-mediated apoptosis of HeLa cells.

    PubMed

    Zhang, Junmin; Li, Ya; Duan, Dongzhu; Yao, Juan; Gao, Kun; Fang, Jianguo

    2016-02-15

    The mammalian thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus, TrxR2 in mitochondria, and TrxR3 in testis, are essential seleno-flavoenzymes with a conserved penultimate selenocysteine (Sec) residue at the C-terminus, and have attracted increasing interests as potential targets for development of cancer chemotherapeutic agents. The sesquiterpene lactone alantolactone (ATL), an active component from the traditional folk medicine Inula helenium, has been documented possessing multiple pharmacological functions, especially the anticancer activity. However, the underlying mechanism has not been well defined. We reported that ATL inhibits both the recombinant TrxR and the enzyme in the cellular environment. The alpha-methylene-gamma-lactone moiety in ATL and the Sec residue in TrxR are critical for targeting TrxR by ATL. By employing our newly developed pull down assay, we demonstrated the remarkable elevation of the oxidized thioredoxin in HeLa cells after ATL treatment. In addition, ATL elicits accumulation of reactive oxygen species, and eventually induces apoptosis of HeLa cells. Importantly, overexpression of the functional TrxR attenuates the cytotoxicity of ATL, while knockdown of the enzyme sensitizes the cells to ATL treatment. Targeting TrxR thus discloses a novel molecular mechanism underlying the cellular action of ATL, and sheds light in considering the usage of ATL as a potential cancer chemotherapeutic agent. PMID:26686580

  11. Influence of seasonal variation and methyl jasmonate mediated induction of glucosinolate biosynthesis on quinone reductase activity in broccoli florets.

    PubMed

    Ku, Kang Mo; Jeffery, Elizabeth H; Juvik, John A

    2013-10-01

    Methyl jasmonate spray treatments (250 μM) were utilized to alter glucosinolate composition in the florets of the commercial broccoli F1 hybrids 'Pirate', 'Expo', 'Green Magic', 'Imperial', and 'Gypsy' grown in replicated field plantings in 2009 and 2010. MeJA treatment significantly increased glucoraphanin (11%), gluconasturtiin (59%), and neoglucobrassicin (248%) concentrations and their hydrolysis products including sulforaphane (152%), phenethyl isothiocyanate (318%), N-methoxyindole-3-carbinol (313%), and neoascorbigen (232%) extracted from florets of these genotypes over two seasons. Increased quinone reductase (QR) activity was significantly correlated with increased levels of sulforaphane, N-methoxyindole-3-carbinol, and neoascorbigen. Partitioning experiment-wide trait variances indicated that the variability in concentrations of sulforaphane (29%), neoascorbigen (48%), and QR activity (72%) was influenced by year-associated weather variables, whereas variation in neoglucobrassicin (63%) and N-methoxyindole-3-carbinol (46%) concentrations was primarily attributed to methyl jasmonate treatment. These results suggest that methyl jasmonate treatment can enhance QR inducing activity by increased hydrolysis of glucoraphanin into sulforaphane and the hydrolysis products of neoglucobrassicin. PMID:24032372

  12. Inhibition of squalene synthase but not squalene cyclase prevents mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis at a posttranscriptional level.

    PubMed

    Peffley, D M; Gayen, A K

    1997-01-15

    Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase

  13. Methionine Sulfoxide Reductase A Negatively Controls Microglia-Mediated Neuroinflammation via Inhibiting ROS/MAPKs/NF-κB Signaling Pathways Through a Catalytic Antioxidant Function

    PubMed Central

    Fan, Hua; Wu, Peng-Fei; Zhang, Ling; Hu, Zhuang-Li; Wang, Wen; Guan, Xin-Lei; Luo, Han; Ni, Ming; Yang, Jing-Wen; Li, Ming-Xing

    2015-01-01

    Abstract Aims: Oxidative burst is one of the earliest biochemical events in the inflammatory activation of microglia. Here, we investigated the potential role of methionine sulfoxide reductase A (MsrA), a key antioxidant enzyme, in the control of microglia-mediated neuroinflammation. Results: MsrA was detected in rat microglia and its expression was upregulated on microglial activation. Silencing of MsrA exacerbated lipopolysaccharide (LPS)-induced activation of microglia and the production of inflammatory markers, indicating that MsrA may function as an endogenous protective mechanism for limiting uncontrolled neuroinflammation. Application of exogenous MsrA by transducing Tat-rMsrA fusion protein into microglia attenuated LPS-induced neuroinflammatory events, which was indicated by an increased Iba1 (a specific microglial marker) expression and the secretion of pro-inflammatory cytokines, and this attenuation was accompanied by inhibiting multiple signaling pathways such as p38 and ERK mitogen-activated protein kinases (MAPKs) and nuclear factor kappaB (NF-κB). These effects were due to MsrA-mediated reactive oxygen species (ROS) elimination, which may be derived from a catalytic effect of MsrA on the reaction of methionine with ROS. Furthermore, the transduction of Tat-rMsrA fusion protein suppressed the activation of microglia and the expression of pro-inflammatory factors in a rat model of neuroinflammation in vivo. Innovation: This study provides the first direct evidence for the biological significance of MsrA in microglia-mediated neuroinflammation. Conclusion: Our data provide a profound insight into the role of endogenous antioxidative defense systems such as MsrA in the control of microglial function. Antioxid. Redox Signal. 22, 832–847. PMID:25602783

  14. Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV.

    PubMed

    Khan, Raees; Lee, Myung Hwan; Joo, Hae-Jin; Jung, Yong-Hoon; Ahmad, Shabir; Choi, Jin-Hee; Lee, Seon-Woo

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria. PMID:25370725

  15. Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA

    PubMed Central

    Koh, Kyung Duk; Balachander, Sathya; Hesselberth, Jay R.; Storici, Francesca

    2015-01-01

    Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed Ribose-seq, a method to capture unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments from yeast Saccharomyces cerevisiae DNA revealed widespread ribonucleotide distribution with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly-synthesized leading strand of nuclear DNA and were present upstream of G+C-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA. PMID:25622106

  16. In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T{sub 3}

    SciTech Connect

    Boone, Lindsey R.; Niesen, Melissa I.; Jaroszeski, Mark; Ness, Gene C.

    2009-07-31

    The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

  17. Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis.

    PubMed

    Chen, Zhong-Hua; Wang, Yizhou; Wang, Jian-Wen; Babla, Mohammad; Zhao, Chenchen; García-Mata, Carlos; Sani, Emanuela; Differ, Christopher; Mak, Michelle; Hills, Adrian; Amtmann, Anna; Blatt, Michael R

    2016-03-01

    Maintaining potassium (K(+) ) nutrition and a robust guard cell K(+) inward channel activity is considered critical for plants' adaptation to fluctuating and challenging growth environment. ABA induces stomatal closure through hydrogen peroxide and nitric oxide (NO) along with subsequent ion channel-mediated loss of K(+) and anions. However, the interactions of NO synthesis and signalling with K(+) nutrition and guard cell K(+) channel activities have not been fully explored in Arabidopsis. Physiological and molecular techniques were employed to dissect the interaction of nitrogen and potassium nutrition in regulating stomatal opening, CO2 assimilation and ion channel activity. These data, gene expression and ABA signalling transduction were compared in wild-type Columbia-0 (Col-0) and the nitrate reductase mutant nia1nia2. Growth and K(+) nutrition were impaired along with stomatal behaviour, membrane transport, and expression of genes associated with ABA signalling in the nia1nia2 mutant. ABA-inhibited K(+) in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for complete stomatal closure in nia1nia2. While NO is an important signalling component in ABA-induced stomatal closure in Arabidopsis, our findings demonstrate a more complex interaction associating potassium nutrition and nitrogen metabolism in the nia1nia2 mutant that affects stomatal function. PMID:26508536

  18. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  19. NasT-Mediated Antitermination Plays an Essential Role in the Regulation of the Assimilatory Nitrate Reductase Operon in Azotobacter vinelandii

    PubMed Central

    Pierson, Leland S.; Rensing, Christopher; Gunatilaka, Malkanthi K.; Kennedy, Christina

    2012-01-01

    Azotobacter vinelandii is a well-studied model system for nitrogen fixation in bacteria. Regulation of nitrogen fixation in A. vinelandii is independent of NtrB/NtrC, a conserved nitrogen regulatory system in proteobacteria. Previous work showed that an ntrC mutation in A. vinelandii resulted in a loss of induction of assimilatory nitrate and nitrite reductases encoded by the nasAB operon. In addition to NtrC, several other proteins, including NasT, a protein containing a potential RNA-binding domain ANTAR (AmiR and NasR transcription antitermination regulators), have been implicated in nasAB regulation. In this work, we characterize the sequence upstream of nasA and identify several DNA sequence elements, including two potential NtrC binding sites and a putative intrinsic transcriptional terminator upstream of nasA that are potentially involved in nasAB regulation. Our analyses confirm that the nasAB promoter, PnasA, is under NtrC control. However, unlike NtrC-regulated promoters in enteric bacteria, PnasA shows high activity in the presence of ammonium; in addition, the PnasA activity is altered in the nifA gene mutation background. We discuss the implication of these results on NtrC-mediated regulation in A. vinelandii. Our study provides direct evidence that induction of nasAB is regulated by NasT-mediated antitermination, which occurs within the leader region of the operon. The results also support the hypothesis that NasT binds the promoter proximal hairpin of nasAB for its regulatory function, which contributes to the understanding of the regulatory mechanism of ANTAR-containing antiterminators. PMID:22773651

  20. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. PMID:26454083

  1. Human DNA Polymerase ϵ Is Able to Efficiently Extend from Multiple Consecutive Ribonucleotides*

    PubMed Central

    Göksenin, A. Yasemin; Zahurancik, Walter; LeCompte, Kimberly G.; Taggart, David J.; Suo, Zucai; Pursell, Zachary F.

    2012-01-01

    Replicative DNA polymerases (Pols) help to maintain the high fidelity of replication in large part through their strong selectivity against mispaired deoxyribonucleotides. It has recently been demonstrated that several replicative Pols from yeast have surprisingly low selectivity for deoxyribonucleotides over their analogous ribonucleotides. In human cells, ribonucleotides are found in great abundance over deoxyribonucleotides, raising the possibility that ribonucleotides are incorporated in the human genome at significant levels during normal cellular functions. To address this possibility, the ability of human DNA polymerase ϵ to incorporate ribonucleotides was tested. At physiological concentrations of nucleotides, human Pol ϵ readily inserts and extends from incorporated ribonucleotides. Almost half of inserted ribonucleotides escape proofreading by 3′ → 5′ exonuclease-proficient Pol ϵ, indicating that ribonucleotide incorporation by Pol ϵ is likely a significant event in human cells. Human Pol ϵ is also efficient at extending from primers terminating in up to five consecutive ribonucleotides. This efficient extension appears to result from reduced exonuclease activity on primers containing consecutive 3′-terminal ribonucleotides. These biochemical properties suggest that Pol ϵ is a likely source of ribonucleotides in human genomic DNA. PMID:23093410

  2. Structural and biochemical characterization of N[superscript 5]-carboxyaminoimidazole ribonucleotide synthetase and N[superscript 5]-carboxyaminoimidazole ribonucleotide mutase from Staphylococcus aureus

    SciTech Connect

    Brugarolas, Pedro; Duguid, Erica M.; Zhang, Wen; Poor, Catherine B.; He, Chuan

    2012-05-08

    With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K{sub m} for bicarbonate was determined for the first time for a PurK enzyme and was found to be {approx}18.8 mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.

  3. TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response

    PubMed Central

    Rozanov, Dmitri; Cheltsov, Anton; Sergienko, Eduard; Vasile, Stefan; Golubkov, Vladislav; Aleshin, Alexander E.; Levin, Trevor; Traer, Elie; Hann, Byron; Freimuth, Julia; Alexeev, Nikita; Alekseyev, Max A.; Budko, Sergey P; Bächinger, Hans Peter; Spellman, Paul

    2015-01-01

    A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model. PMID:26075913

  4. TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response.

    PubMed

    Rozanov, Dmitri; Cheltsov, Anton; Sergienko, Eduard; Vasile, Stefan; Golubkov, Vladislav; Aleshin, Alexander E; Levin, Trevor; Traer, Elie; Hann, Byron; Freimuth, Julia; Alexeev, Nikita; Alekseyev, Max A; Budko, Sergey P; Bächinger, Hans Peter; Spellman, Paul

    2015-01-01

    A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model. PMID:26075913

  5. Regulation of Folate-Mediated One-Carbon Metabolism by Glycine N-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR).

    PubMed

    Wang, Yi-Cheng; Wu, Ming-Tsung; Lin, Yan-Jun; Tang, Feng-Yao; Ko, Hsin-An; Chiang, En-Pei

    2015-01-01

    Folate-mediated one-carbon metabolism is an important therapeutic target of human diseases. We extensively investigated how gene-nutrient interactions may modulate human cancer risk in 2 major folate metabolic genes, MTHFR and GNMT. The biochemical impacts of MTHFR and GNMT on methyl group supply, global DNA methylation, nucleotide biosynthesis, DNA damage, and partitioning of the folate dependent 1-carbon group were carefully studied. The distinct model systems used included: EB virus-transformed lymphoblasts expressing human MTHFR polymorphic genotypes; liver-derived GNMT-null cell-lines with and without GNMT overexpression; and HepG2 cells with stabilized inhibition of MTHFR using shRNA, GNMT wildtype, heterozygotous (GNMT(het)) and knockout (GNMT(nul)) mice. We discovered that the MTHFR TT genotype significantly reduces folate-dependent remethylation under folate restriction, but it assists purine synthesis when folate is adequate. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in human hematological malignancies. GNMT affects transmethylation kinetics and S-adenosylmethionine (adoMet) synthesis, and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. Restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. The systematic series of studies gives new insights into the underlying mechanisms by which MTHFR and GNMT may participate in human tumor prevention. PMID:26598833

  6. The role of aldo-keto reductase 1C3 (AKR1C3)-mediated prostaglandin D2 (PGD2) metabolism in keloids.

    PubMed

    Mantel, Alon; Newsome, Austin; Thekkudan, Theresa; Frazier, Robert; Katdare, Meena

    2016-01-01

    Keloids are progressively expanding scars, mostly prevalent in individuals of African descent. Previous data identified increased mast cell number and activation state in keloids suggesting a role in disease progression. The major eicosanoid secreted by mast cells is prostaglandin D2 (PGD2), a relatively unstable pro-inflammatory mediator which can be spontaneously converted to 15-deoxy-(Delta12,14)-prostaglandin J2(15d-PGJ2) or enzymatically metabolized to 9α,11β-PGF2 by aldo-keto reductase 1C3 (AKR1C3). In this work, we investigated the possible role of PGD2 and its metabolites in keloids using CRL1762 keloid fibroblasts (KF) and immunohistochemical staining. Our data suggested approximately 3-fold increase of tryptase-positive mast cell count in keloids compared with normal skin. Furthermore, AKR1C3 was overexpressed in the fibrotic area of keloids while relatively weak staining detected in normal skin. Metabolism of PGD2 to 9α,11β-PGF2 by both, KF and normal fibroblasts, was dependent on AKR1C3 as this reaction was attenuated in the presence of the AKR1C3 inhibitor, 2'-hydroxyflavanone, or in cells with decreased AKR1C3 expression. 15d-PGJ2, but not the other tested PGs, inhibited KF proliferation, attenuated KF-mediated collagen gel contraction and increased caspase-3 activation. In addition, treatment with 15d-PGJ2 activated P38-MAPK, induced reactive oxygen species and upregulated superoxide dismutase-1 (SOD-1). Finally, inhibition of P38-MAPK further augmented 15d-PGJ2-induced caspase-3 cleavage and attenuated its effect on SOD-1 transcription. This work suggests that localized dual inhibition of AKR1C3 and P38-MAPK may inhibit keloid progression. Inhibiting AKR1C3 activity may generate oxidative environment due to redirection of PGD2 metabolism towards 15d-PGJ2 while inhibition of P38-MAPK will sensitize keloid cells to ROS-induced apoptosis. PMID:26308156

  7. Structure-function analysis of ribonucleotide bypass by B family DNA replicases

    SciTech Connect

    Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.; Pedersen, Lars C.; Kunkel, Thomas A.

    2013-11-01

    Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.

  8. Subacute methotrexate neurotoxicity and cerebral venous sinus thrombosis in a 12-year-old with acute lymphoblastic leukemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: homocysteine-mediated methotrexate neurotoxicity via direct endothelial injury.

    PubMed

    Mahadeo, Kris M; Dhall, Girish; Panigrahy, Ashok; Lastra, Carlos; Ettinger, Lawrence J

    2010-02-01

    From as early as the 1970s methotrexate has been associated with disseminated necrotizing leukoencephalopathy and other neurotoxic sequelae. Yet, a clear mechanism for methotrexate-induced neurotoxicity has not been established. The authors describe the case of a 12-year-old male with acute lymphoblastic leukemia and a homozygous methylenetetrahydrofolate reductase C677T mutation, who developed subacute methotrexate-induced toxicity and cerebral venous thrombosis after receiving intrathecal methotrexate. The role of homocysteine as a possible mediator in methotrexate-induced neurotoxicity via direct endothelial injury is discussed. PMID:20121554

  9. Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima: Structural Insights into Complex Formation

    SciTech Connect

    Morar, Mariya; Hoskins, Aaron A.; Stubbe, JoAnne; Ealick, Steven E.

    2008-10-02

    In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, P{sub i}, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified.

  10. Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

    PubMed

    Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

    2015-10-23

    The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. PMID:26338705

  11. [Interaction of Ag+ ions with ribonucleotides of canonical bases].

    PubMed

    Sorokin, V A; Valeev, V A; Gladchenko, G O; Sysa, I V; Degtiar, M V; Volchok, I V; Blagoĭ, Iu P

    1999-01-01

    The interaction of Ag+ ions with ribonucleotides of canonical bases in aqueous solution was studied by differential UV spectroscopy. Atoms coordinating silver ions (N7, O6 of guanosine 5'-monophosphate, N3, O2 of cytidine 5'-monophosphate, N7, N1, N3 of adenosine 5'-monophosphate and N3 of uridine 5'-monophosphate) and the binding constants characterizing the formation of appropriate complexes were determined. The differences in the relative affinity of Ag+ ions for the atoms of nucleotide bases correlate with the potential on them. PMID:10418671

  12. Investigating the intermediates in the reaction of ribonucleoside triphosphate reductase from Lactobacillus leichmannii : An application of HF EPR-RFQ technology

    NASA Astrophysics Data System (ADS)

    Manzerova, Julia; Krymov, Vladimir; Gerfen, Gary J.

    2011-12-01

    In this investigation high-frequency electron paramagnetic resonance spectroscopy (HFEPR) in conjunction with innovative rapid freeze-quench (RFQ) technology is employed to study the exchange-coupled thiyl radical-cob(II)alamin system in ribonucleotide reductase from a prokaryote Lactobacillus leichmannii. The size of the exchange coupling ( Jex) and the values of the thiyl radical g tensor are refined, while confirming the previously determined (Gerfen et al. (1996) [20]) distance between the paramagnets. Conclusions relevant to ribonucleotide reductase catalysis and the architecture of the active site are presented. A key part of this work has been the development of a unique RFQ apparatus for the preparation of millisecond quench time RFQ samples which can be packed into small (0.5 mm ID) sample tubes used for CW and pulsed HFEPR - lack of this ability has heretofore precluded such studies. The technology is compatible with a broad range of spectroscopic techniques and can be readily adopted by other laboratories.

  13. Importance of the Maintenance Pathway in the Regulation of the Activity of Escherichia coli Ribonucleotide Reductase†

    PubMed Central

    2008-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α2β2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on α. [Y•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an l-arabinose promoter were constructed. Using these constructs and with [l-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 µM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y• in vivo in E. coli is a mechanism of regulating RNR activity. PMID:18314964

  14. Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase*

    PubMed Central

    Kasiviswanathan, Rajesh; Copeland, William C.

    2011-01-01

    During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2′-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3–14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3′-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed. PMID:21778232

  15. Solution Structure of the Dickerson DNA Dodecamer Containing a Single Ribonucleotide

    PubMed Central

    DeRose, Eugene F.; Perera, Lalith; Murray, Michael S.; Kunkel, Thomas A.; London, Robert E.

    2013-01-01

    Ribonucleotides are frequently incorporated into DNA during replication. They are recognized and processed by several cellular enzymes, and their continued presence in the yeast nuclear genome results in replicative stress and genome instability. Thus, it is important to understand the effects of isolated ribonucleotide incorporation on DNA structure. Towards this goal, here we describe the NMR structure of the self-complementary Dickson dodecamer sequence [d(CGC)rGd(AATTCGCG)]2 containing two symmetrically positioned riboguanosines. The absence of an observable H1-H2 scalar coupling interaction indicates a C3'-endo conformation for the ribose. Longer-range structural perturbations resulting from the presence of the ribonucleotide are limited to the adjacent and trans-helical nucleotides, while the global B-form DNA structure is maintained. Since crystallographic studies have indicated that isolated ribonucleotides promote global B→A transitions, we also performed molecular modeling analyses to evaluate the structural consequences of higher ribonucleotide substitution levels. Increasing the ribonucleotide content increased the minor groove width toward values more similar to A-DNA, but even 50% ribonucleotide substitution did not fully convert the B-DNA to A-DNA. Comparing the present structure with the structure of an RNase H2-bound DNA supports the conclusion that, as with other DNA-protein complexes, the DNA conformation is strongly influenced by the interaction with the protein. PMID:22390730

  16. Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

    PubMed Central

    Günther, Claudia; Kind, Barbara; Reijns, Martin A.M.; Berndt, Nicole; Martinez-Bueno, Manuel; Wolf, Christine; Tüngler, Victoria; Chara, Osvaldo; Lee, Young Ae; Hübner, Norbert; Bicknell, Louise; Blum, Sophia; Krug, Claudia; Schmidt, Franziska; Kretschmer, Stefanie; Koss, Sarah; Astell, Katy R.; Ramantani, Georgia; Bauerfeind, Anja; Morris, David L.; Cunninghame Graham, Deborah S.; Bubeck, Doryen; Leitch, Andrea; Ralston, Stuart H.; Blackburn, Elizabeth A.; Gahr, Manfred; Witte, Torsten; Vyse, Timothy J.; Melchers, Inga; Mangold, Elisabeth; Nöthen, Markus M.; Aringer, Martin; Kuhn, Annegret; Lüthke, Kirsten; Unger, Leonore; Bley, Annette; Lorenzi, Alice; Isaacs, John D.; Alexopoulou, Dimitra; Conrad, Karsten; Dahl, Andreas; Roers, Axel; Alarcon-Riquelme, Marta E.; Jackson, Andrew P.; Lee-Kirsch, Min Ae

    2014-01-01

    Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2–associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage–associated pathways in the initiation of autoimmunity. PMID:25500883

  17. Darwinian behavior in a cold, sporadically fed pool of ribonucleotides.

    PubMed

    Yarus, Michael

    2012-09-01

    A testable, explicit origin for Darwinian behavior, feasible on a chaotic early Earth, would aid origins discussion. Here I show that a pool receiving unreliable supplies of unstable ribonucleotide precursors can recurrently fill this role. By using numerical integration, the differential equations governing a sporadically fed pool are solved, yielding quantitative constraints for the proliferation of molecules that also have a chemical phenotype. For example, templated triphosphate nucleotide joining is >10(4) too slow, suggesting that a group more reactive than pyrophosphate activated primordial nucleotides. However, measured literature rates are sufficient if the Initial Darwinian Ancestor (IDA) resembles a 5'-5' cofactor-like dinucleotide RNA, synthesized via activation with a phosphorimidazolide-like group. A sporadically fed pool offers unforeseen advantages; for example, the pool hosts a novel replicator which is predominantly unpaired, even though it replicates. Such free template is optimized for effective selection during its replication. Pool nucleotides are also subject to a broadly based selection that impels the population toward replication, effective selection, and Darwinian behavior. Such a primordial pool may have left detectable modern traces. A sporadically fed ribonucleotide pool also fits a recognizable early Earth environment, has recognizable modern descendants, and suits the early shape of the phylogenetic tree of Earthly life. Finally, analysis points to particular data now needed to refine the hypothesis. Accordingly, a kinetically explicit chemical hypothesis for a terran IDA can be justified, and informative experiments seem readily accessible. PMID:22946838

  18. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  19. Darwinian Behavior in a Cold, Sporadically Fed Pool of Ribonucleotides

    PubMed Central

    2012-01-01

    Abstract A testable, explicit origin for Darwinian behavior, feasible on a chaotic early Earth, would aid origins discussion. Here I show that a pool receiving unreliable supplies of unstable ribonucleotide precursors can recurrently fill this role. By using numerical integration, the differential equations governing a sporadically fed pool are solved, yielding quantitative constraints for the proliferation of molecules that also have a chemical phenotype. For example, templated triphosphate nucleotide joining is >104 too slow, suggesting that a group more reactive than pyrophosphate activated primordial nucleotides. However, measured literature rates are sufficient if the Initial Darwinian Ancestor (IDA) resembles a 5′-5′ cofactor-like dinucleotide RNA, synthesized via activation with a phosphorimidazolide-like group. A sporadically fed pool offers unforeseen advantages; for example, the pool hosts a novel replicator which is predominantly unpaired, even though it replicates. Such free template is optimized for effective selection during its replication. Pool nucleotides are also subject to a broadly based selection that impels the population toward replication, effective selection, and Darwinian behavior. Such a primordial pool may have left detectable modern traces. A sporadically fed ribonucleotide pool also fits a recognizable early Earth environment, has recognizable modern descendants, and suits the early shape of the phylogenetic tree of Earthly life. Finally, analysis points to particular data now needed to refine the hypothesis. Accordingly, a kinetically explicit chemical hypothesis for a terran IDA can be justified, and informative experiments seem readily accessible. Key Words: Cofactor—RNA—Origin of life—Replication—Initial Darwinian Ancestor (IDA). Astrobiology 12, 870–883. PMID:22946838

  20. In Situ Imidazole Activation of Ribonucleotides for Abiotic RNA Oligomerization Reactions

    NASA Astrophysics Data System (ADS)

    Burcar, Bradley T.; Jawed, Mohsin; Shah, Hari; McGown, Linda B.

    2015-06-01

    The hypothesis that RNA played a significant role in the origin of life requires effective and efficient abiotic pathways to produce RNA oligomers. The most successful abiotic oligomerization reactions to date have utilized high-energy, modified, or pre-activated ribonucleotides to generate strands of RNA up to 50-mers in length. In spite of their success, these modifications and pre-activation reactions significantly alter the ribonucleotides in ways that are highly unlikely to have occurred on a prebiotic Earth. This research seeks to address this problem by exploring an aqueous based method for activating the canonical ribonucleotides in situ using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. The reactions were run with and without a montmorillonite clay catalyst and compared to reactions that used ribonucleotides that were pre-activated with imidazole. The effects of pH and ribonucleotide concentration were also investigated. The results demonstrate the ability of in situ activation of ribonucleotides to generate linear RNA oligomers in solution, providing an alternative route to produce RNA for use in prebiotic Earth scenarios.

  1. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

    PubMed Central

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  2. Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2.

    PubMed

    Chang, Emily Yun-Chia; Chang, Yi-Cheng; Shun, Chia-Tung; Tien, Yu-Wen; Tsai, Shu-Huei; Hee, Siow-Wey; Chen, Ing-Jung; Chuang, Lee-Ming

    2016-01-01

    Prostaglandin reductase 2 (PTGR2) is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), two important providers of intracellular cysteine for the generation of glutathione (GSH), which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer. PMID:26820738

  3. Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2

    PubMed Central

    Chang, Emily Yun-Chia; Chang, Yi-Cheng; Shun, Chia-Tung; Tien, Yu-Wen; Tsai, Shu-Huei; Hee, Siow-Wey; Chen, Ing-Jung; Chuang, Lee-Ming

    2016-01-01

    Prostaglandin reductase 2 (PTGR2) is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), two important providers of intracellular cysteine for the generation of glutathione (GSH), which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer. PMID:26820738

  4. RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: consequences on lignans and neolignans accumulation.

    PubMed

    Renouard, Sullivan; Tribalatc, Marie-Aude; Lamblin, Frederic; Mongelard, Gaëlle; Fliniaux, Ophélie; Corbin, Cyrielle; Marosevic, Djurdjica; Pilard, Serge; Demailly, Hervé; Gutierrez, Laurent; Hano, Christophe; Mesnard, François; Lainé, Eric

    2014-09-15

    RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5' linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds. PMID:25046758

  5. Flavodoxin-mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena: role of flavodoxin hydrophobic residues in protein-protein interactions.

    PubMed

    Goñi, Guillermina; Serrano, Ana; Frago, Susana; Hervás, Manuel; Peregrina, José Ramón; De la Rosa, Miguel A; Gómez-Moreno, Carlos; Navarro, José A; Medina, Milagros

    2008-01-29

    Three surface hydrophobic residues located at the Anabaena flavodoxin (Fld) putative complex interface with its redox partners were replaced by site-directed mutagenesis. The effects of these replacements on Fld interaction with both its physiological electron donor, photosystem I (PSI), and its electron acceptor, ferredoxin-NADP+ reductase (FNR), were analyzed. Trp57, Ile59, and Ile92 contributed to the optimal orientation and tightening of the FNR:Fld and PSI:Fld complexes. However, these side chains did not appear to be involved in crucial specific interactions, but rather contributed to the obtainment of the optimal orientation and distance of the redox centers required for efficient electron transfer. This supports the idea that the interaction of Fld with its partners is less specific than that of ferredoxin and that more than one orientation is efficient for electron transfer in these transient complexes. Additionally, for some of the analyzed processes, WT Fld seems not to be the most optimized molecular species. Therefore, subtle changes at the isoalloxazine environment not only influence the Fld binding abilities, but also modulate the electron exchange processes by producing different orientations and distances between the redox centers. Finally, the weaker apoflavodoxin interaction with FNR suggests that the solvent-accessible region of FMN plays a role either in complex formation with FNR or in providing the adequate conformation of the FNR binding region in Fld. PMID:18177021

  6. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  7. The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ

    PubMed Central

    Gosavi, Rajendrakumar A.; Moon, Andrea F.; Kunkel, Thomas A.; Pedersen, Lars C.; Bebenek, Katarzyna

    2012-01-01

    Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2′-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein–nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high. PMID:22584622

  8. Analysis of cytochrome b(5) reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence.

    PubMed

    Derbyshire, Mark C; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-09-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC-MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  9. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly*

    PubMed Central

    Askenasy, Isabel; Pennington, Joseph M.; Tao, Yeqing; Marshall, Alan G.; Young, Nicolas L.; Shang, Weifeng; Stroupe, M. Elizabeth

    2015-01-01

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme. PMID:26088143

  10. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence

    PubMed Central

    Derbyshire, Mark C.; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J.; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-01-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC–MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  11. [Methylenetetrahydrofolate reductase polymorphism C677T in patients with consolidated fractures and pseudarthrosis of long bones: relationship with homocystein and inflammatory mediators].

    PubMed

    Bezsmertnyĭ, Iu O

    2013-01-01

    In article described research the results of the prevalence of the genetic polymorphism of the gene Methylentetrahydrofolatereductase C677T (MTHFR) in 130 patients with pseudarthrosis of long bones and in those with consolidated fractures. The incidence of allele-T among patients with pseudarthrosis was 1.4 times higher than among those with consolidated fractures. Pathological genotype MTHFR 677-TT was associated with the development avital types of pseudarthrosis and increase the proportion of people with hyperhomocysteinemia, high content of inflammatory mediators and development refracture. PMID:24605633

  12. Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Huston, Donald; Kuban, Wojciech; Liu, Lili; Van Houten, Bennett; Woodgate, Roger

    2013-01-01

    Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the

  13. Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit.

    PubMed

    Meurisse, Julie; Bacquin, Agathe; Richet, Nicolas; Charbonnier, Jean-Baptiste; Ochsenbein, Françoise; Peyroche, Anne

    2014-12-01

    Rad53 is a conserved protein kinase with a central role in DNA damage response and nucleotide metabolism. We observed that the expression of a dominant-lethal form of RAD53 leads to significant expression changes for at least 16 genes, including the RNR3 and the HUG1 genes, both of which are involved in the control of nucleotide metabolism. We established by multiple biophysical and biochemical approaches that Hug1 is an intrinsically disordered protein that directly binds to the small RNR subunit Rnr2. We characterized the surface of interaction involved in Hug1 binding to Rnr2, and we thus defined a new binding region to Rnr2. Moreover, we show that Hug1 is deleterious to cell growth in the context of reduced RNR activity. This inhibitory effect of Hug1 on RNR activity depends on the binding of Hug1 to Rnr2. We propose a model in which Hug1 modulates Rnr2-Rnr1 association by binding Rnr2. We show that Hug1 accumulates under various physiological conditions of high RNR induction. Hence, both the regulation and the mode of action of Hug1 are different from those of the small protein inhibitors Dif1 and Sml1, and Hug1 can be considered as a regulator for fine-tuning of RNR activity. PMID:25378334

  14. S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata

    PubMed Central

    Wünsche, Hendrik; Baldwin, Ian T.; Wu, Jianqiang

    2011-01-01

    S-nitrosoglutathione reductase (GSNOR) reduces the nitric oxide (NO) adduct S-nitrosoglutathione (GSNO), an essential reservoir for NO bioactivity. In plants, GSNOR has been found to be important in resistance to bacterial and fungal pathogens, but whether it is also involved in plant–herbivore interactions was not known. Using a virus-induced gene silencing (VIGS) system, the activity of GSNOR in a wild tobacco species, Nicotiana attenuata, was knocked down and the function of GSNOR in defence against the insect herbivore Manduca sexta was examined. Silencing GSNOR decreased the herbivory-induced accumulation of jasmonic acid (JA) and ethylene, two important phytohormones regulating plant defence levels, without compromising the activity of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Decreased activity of trypsin proteinase inhibitors (TPIs) were detected in GSNOR-silenced plants after simulated M. sexta feeding and bioassays indicated that GSNOR-silenced plants have elevated susceptibility to M. sexta attack. Furthermore, GSNOR is required for methyl jasmonate (MeJA)-induced accumulation of defence-related secondary metabolites (TPI, caffeoylputrescine, and diterpene glycosides) but is not needed for the transcriptional regulation of JAZ3 (jasmonate ZIM-domain 3) and TD (threonine deaminase), indicating that GSNOR mediates certain but not all jasmonate-inducible responses. This work highlights the important role of GSNOR in plant resistance to herbivory and jasmonate signalling and suggests the potential involvement of NO in plant–herbivore interactions. Our data also suggest that GSNOR could be a target of genetic modification for improving crop resistance to herbivores. PMID:21622839

  15. Hydroxyurea induces hydroxyl radical-mediated cell death in Escherichia coli

    PubMed Central

    Davies, Bryan W.; Kohanski, Michael A.; Simmons, Lyle A.; Winkler, Jonathan A.; Collins, James J.; Walker, Graham C.

    2010-01-01

    SUMMARY Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. While HU inhibition of RNR has been recognized for decades, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability in the majority of the cell population. Continued HU stress activates iron uptake as well as the toxins MazF and RelE whose activity causes the synthesis of incompletely translated proteins and stimulation of the envelope stress response system. These effects alter the properties of one of the cell’s two terminal cytochrome oxidases in the electron transport chain, causing an increase in the production of superoxide. The increased superoxide production from the respiratory chain together with the increased iron uptake fuels the formation of hydroxyl radicals that contribute to HU-induced cell death. This work significantly expands our understanding of HU-mediated cell death and more broadly suggests a pathway whereby replication fork arrest leads to cell death. PMID:20005847

  16. A Mn(IV)/Fe(IV) Intermediate in Assembly of the Mn(IV)/Fe(III) Cofactor of Chlamydia trachomatis Ribonucleotide Reductase†

    PubMed Central

    Jiang, Wei; Hoffart, Lee M.; Krebs, Carsten; Bollinger, J. Martin

    2008-01-01

    We recently showed that the class Ic ribonucleotide reductase from the human pathogen, Chlamydia trachomatis, uses a MnIV/FeIII cofactor to generate protein and substrate radicals in its catalytic mechanism [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the MnII/FeII cluster and O2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active MnIV/FeIII-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than MnIV/FeIII. Mössbauer spectra show that the intermediate contains a high-spin FeIV center. Its chemical and spectroscopic properties establish that the intermediate is a MnIV/FeIV-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the MnIV (SMn = 3/2) and high-spin FeIV (SFe = 2) sites. PMID:17616152

  17. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed Central

    Sengupta, S; Shaila, M S; Rao, G R

    1996-01-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems. PMID:8694757

  18. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA.

    PubMed Central

    Batey, R T; Inada, M; Kujawinski, E; Puglisi, J D; Williamson, J R

    1992-01-01

    A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR. Images PMID:1383928

  19. Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Joyce, Gerald F.

    2001-01-01

    A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the non-template and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and P-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that employ T7 RNA polymerase for in vitro transcription.

  20. Small interference RNA-mediated gene silencing of human biliverdin reductase, but not that of heme oxygenase-1, attenuates arsenite-mediated induction of the oxygenase and increases apoptosis in 293A kidney cells.

    PubMed

    Miralem, Tihomir; Hu, Zhenbo; Torno, Michael D; Lelli, Katherine M; Maines, Mahin D

    2005-04-29

    BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4-5-fold) after treatment of 293A cells with 5 mum As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 mum As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation. PMID:15741166

  1. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-08-01

    Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions. PMID:6778961

  2. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  3. Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic Mismatch Repair

    PubMed Central

    Ghodgaonkar, Medini Manohar; Lazzaro, Federico; Olivera-Pimentel, Maite; Artola-Borán, Mariela; Cejka, Petr; Reijns, Martin A.; Jackson, Andrew P.; Plevani, Paolo; Muzi-Falconi, Marco; Jiricny, Josef

    2013-01-01

    Summary To improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand. PMID:23603115

  4. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  5. The Sterol-sensing Domain (SSD) Directly Mediates Signal-regulated Endoplasmic Reticulum-associated Degradation (ERAD) of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Isozyme Hmg2*

    PubMed Central

    Theesfeld, Chandra L.; Pourmand, Deeba; Davis, Talib; Garza, Renee M.; Hampton, Randolph Y.

    2011-01-01

    The sterol-sensing domain (SSD) is a conserved motif in membrane proteins responsible for sterol regulation. Mammalian proteins SREBP cleavage-activating protein (SCAP) and HMG-CoA reductase (HMGR) both possess SSDs required for feedback regulation of sterol-related genes and sterol synthetic rate. Although these two SSD proteins clearly sense sterols, the range of signals detected by this eukaryotic motif is not clear. The yeast HMG-CoA reductase isozyme Hmg2, like its mammalian counterpart, undergoes endoplasmic reticulum (ER)-associated degradation that is subject to feedback control by the sterol pathway. The primary degradation signal for yeast Hmg2 degradation is the 20-carbon isoprene geranylgeranyl pyrophosphate, rather than a sterol. Nevertheless, the Hmg2 protein possesses an SSD, leading us to test its role in feedback control of Hmg2 stability. We mutated highly conserved SSD residues of Hmg2 and evaluated regulated degradation. Our results indicated that the SSD was required for sterol pathway signals to stimulate Hmg2 ER-associated degradation and was employed for detection of both geranylgeranyl pyrophosphate and a secondary oxysterol signal. Our data further indicate that the SSD allows a signal-dependent structural change in Hmg2 that promotes entry into the ER degradation pathway. Thus, the eukaryotic SSD is capable of significant plasticity in signal recognition or response. We propose that the harnessing of cellular quality control pathways to bring about feedback regulation of normal proteins is a unifying theme for the action of all SSDs. PMID:21628456

  6. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  7. NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum dimerizes at high protein concentration and exclusively receives electrons from thioredoxin reductase.

    PubMed

    Van Laer, Koen; Dziewulska, Aleksandra M; Fislage, Marcus; Wahni, Khadija; Hbeddou, Abderahim; Collet, Jean-Francois; Versées, Wim; Mateos, Luis M; Tamu Dufe, Veronica; Messens, Joris

    2013-03-15

    NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10(6) and 5.6 × 10(6) M(-1) min(-1), respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pKa lower than 6.5 for nucleophilic attack, whereas the pKa of the C-terminal cysteine is ~10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum. PMID:23362277

  8. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  9. Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms.

    PubMed

    Firestine, S M; Poon, S W; Mueller, E J; Stubbe, J; Davisson, V J

    1994-10-01

    A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7918411

  10. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  11. Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair

    PubMed Central

    Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni

    2016-01-01

    Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111

  12. Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics.

    PubMed

    Rose, Rebecca E; Quinn, Ryan; Sayre, Jackie L; Fabris, Daniele

    2015-07-01

    The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS(n)) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance. PMID:25995446

  13. Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics

    PubMed Central

    Rose, Rebecca E.; Quinn, Ryan; Sayre, Jackie L.

    2015-01-01

    The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MSn) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance. PMID:25995446

  14. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

  15. Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope.

    PubMed

    Zaumseil, J; Wittstock, G; Bahrs, S; Steinrücke, P

    2000-06-01

    Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator. PMID:11225859

  16. A phase I pharmacodynamic study of GTI-2040, an antisense oligonucleotide against ribonuclotide reductase, in acute leukemias: a California Cancer Consortium study.

    PubMed

    Kirschbaum, Mark H; Frankel, Paul; Synold, Timothy W; Xie, Zhiliang; Yen, Yun; Popplewell, Leslie; Chen, Robert; Aljitawi, Omar; Tuscano, Joseph M; Chan, Kenneth K; Newman, Edward M

    2016-10-01

    We performed a phase I study of GTI-2040, an antisense oligonucleotide against ribonucleotide reductase mRNA, on a novel dosing schedule of days 1-4 and 15-18 by continuous infusion to examine efficacy and tolerability in patients with leukemia. A dose of 11 mg/kg/d was safely reached. Dose-limiting toxicities (DLTs) at the higher levels included elevated troponin I and liver function enzymes. There were no objective responses to GTI-2040 in this study; 7/24 patients were able to complete the predetermined three infusion cycles. Pharmacokinetic and pharmacodynamic studies were performed, indicating a trend towards increasing intracellular drug levels and decreasing RRM2 gene expression with increasing doses. This dose schedule may be considered if appropriate combinations are identified in preclinical studies. PMID:26895565

  17. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  18. Nuclear magnetic resonance studies of 2'- and 3'-ribonucleotide structures in solution.

    PubMed

    Davies, D B; Danyluk, S S

    1975-02-11

    A systematic 220-MHz proton nuclear magnetic resonance (nmr) study has been made of all common purine and pyrimidine 2'(3')-ribonucleotides in D2O solutions at 20 plus or minus 2 degrees. Spectra for the entire series were measured under similar conditions of concentration, temperature, and ionic strength, thereby facilitating intercomparisons of spectral properties. Spectral assignments were accomplished with the aid of selected 31P-1H decoupling experiments and accurate values of nmr parameters were derived by simulation-iteration procedures. A detailed analysis of the coupling constants and chemical shifts permitted a determination of conformational properties for ribose rings, exocyclic carbinol and phosphate groups, and the orientation of base-ribose rings. Following procedures described elsewhere, an evaluation was made of ribose ring pseudorotational parameters for each 2'(3')-nucleotide. The results show that both the degree of pucker and pseudorotational angle vary only slightly throughout the entire series of molecules, and lie within the ranges found in the crystalline state. Furthermore, the ribose rings are in rapid equilibrium between N type (C(3')-ENDO, C(2')-exo) and S type (C(2')-endo, C(3')-exo) conformers, N forms and is formed by S, with an S type conformer favored in purine 2'(3')-ribonucleotides (-60:40) while pyrimidines exhibit approximately equal compositions. Thus, the phosphate location on the ring has less of an effect on ring properties than the nature of the base ring. An analysis is also reported of rotamer equilibria about C(4')-C(5'), C(2')-O(2'), and C(3')-O(3') bonds. For the former the nmr coupling constant data are consistent with a predominant gg rotamer (-70%) with gt and tg rotamers populated to the extent of -20 and -10%, respectively. No correlation of the type seen for 5'-nucleotides appears to exist between C(4')-C(5') gg population and ribose ring equilibrium composition. For 2'-nucleotides the 31P-H(2') coupling data

  19. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism. PMID:26746798

  20. Using chemical approaches to study selenoproteins - focus on thioredoxin reductases

    PubMed Central

    Hondal, Robert J.

    2009-01-01

    The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine. PMID:19406205

  1. Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

    PubMed Central

    Ding, James; Taylor, Martin S.; Jackson, Andrew P.; Reijns, Martin A. M.

    2016-01-01

    Ribonucleotides are the most common non-canonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by Ribonuclease (RNase) H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae we developed embedded Ribonucleotide Sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3’-hydroxyl groups, in contrast to alternative methods that utilize alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. This protocol can be adapted for the genome-wide mapping of other non-canonical bases by replacing RNase H2 with specific nicking endonucleases, a method we term Endonuclease Sequencing (EndoSeq). With the protocol taking <5 days to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions. PMID:26313479

  2. Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines.

    PubMed Central

    Minasov, G; Matulic-Adamic, J; Wilds, C J; Haeberli, P; Usman, N; Beigelman, L; Egli, M

    2000-01-01

    Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs. PMID:11105752

  3. Crystal Structure and Function of 5-Formaminoimidazole-4-carboxamide Ribonucleotide Synthetase from Methanocaldococcus jannaschii

    SciTech Connect

    Zhang, Yang; White, Robert H.; Ealick, Steven E.

    2008-08-06

    Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.

  4. The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase

    SciTech Connect

    Liu, M.-C.; Peck, H.D., Jr.

    1981-12-01

    Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal M/sub r/=66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced..cap alpha..-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase,however, is able to transfer electrons from H/sub 2/ to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially reoxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57/sup o/C. Purified nitrite reductase also has hydroxylamine reductase activity, and the K/sub m/ for nitrite was determined to be 1.14 mM.

  5. Dihydropteridine reductase from Escherichia coli.

    PubMed Central

    Vasudevan, S G; Shaw, D C; Armarego, W L

    1988-01-01

    A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor. Images Fig. 2. PMID:3060113

  6. Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits o...

  7. Phase I study of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) in combination with high dose cytarabine in patients with advanced myeloid leukemia

    PubMed Central

    Larson, Richard A.; Gajria, Devika; Dolan, M. Eileen; Delaney, Shannon M.; Karrison, Theodore G.; Ratain, Mark J.; Stock, Wendy

    2014-01-01

    Summary Purpose This Phase I dose escalation study was based on the hypothesis that the addition of 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) to cytarabine would enhance cytarabine cytotoxicity. The primary objective of the study was to establish the maximum tolerated dose of 3-AP when given in combination with a fixed dose of cytarabine. Experimental design Twenty-five patients with relapsed or refractory myeloid leukemia were enrolled to three dose levels of 3-AP. Cytarabine was administered as a 2 h infusion at a fixed dose of 1,000 mg/m2/day for 5 consecutive days. Escalating doses of 3-AP as a 2 h infusion were administered on days 2 through 5. The 3-AP infusion preceded the start of the cytarabine infusion by 4 h. Results In general, the toxicities observed with the combination were similar to the expected toxicity profile for cytarabine when utilized as a single agent at this dose and schedule. However, two of three patients developed dose-limiting methemoglobinemia at the highest 3-AP dose studied (100 mg/m2). Transient reversible methemoglobinemia was documented in 11 of 15 patients enrolled at the 75 mg/m2 dose level. Objective evidence of clinical activity was observed in four patients. Conclusions The combination of 3-AP and cytarabine given on this schedule is feasible in advanced myeloid leukemia. The recommended Phase II dose is 75 mg/m2/day of 3-AP on days 2–5 given prior to cytarabine administered at a dose of 1,000 mg/m2/day over 5 consecutive days. Methemoglobinemia is a common toxicity of this combination and requires close monitoring. PMID:18217206

  8. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. PMID:20561902

  9. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  10. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. ); Malfoy, B. ); Forrest, G.L. )

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.