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Sample records for 2-5a synthetase induction

  1. Sponge OAS has a distinct genomic structure within the 2-5A synthetase family.

    PubMed

    Reintamm, Tõnu; Kuusksalu, Anne; Metsis, Madis; Päri, Mailis; Vallmann, Kerli; Lopp, Annika; Justesen, Just; Kelve, Merike

    2008-11-01

    2',5'-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family.

  2. Detection of elevated levels of 2-5A synthetase in serum from children with various infectious diseases.

    PubMed Central

    Sugino, H; Mitani, I; Koike, M; Kodama, T; Sokawa, J; Sawai, H; Ishibashi, K; Itoh, M; Watanabe, S; Sokawa, Y

    1986-01-01

    By a sensitive radioimmunoassay method, (2'-5')oligoadenylate synthetase was detected in serum from patients with viral, bacterial, or mycoplasmal infections at elevated levels compared with enzyme levels in serum from healthy individuals and patients suffering from noninfectious diseases. PMID:3760142

  3. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  4. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  5. Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger.

    PubMed

    Sealy-Lewis, H M; Fairhurst, V

    1998-07-01

    Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

  6. Cell contacts are required for induction by cortisol of glutamine synthetase gene transcription in the retina.

    PubMed Central

    Vardimon, L; Fox, L L; Degenstein, L; Moscona, A A

    1988-01-01

    In embryonic neural retina the enzyme glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] is a glia-specific differentiation marker inducible with cortisol. We show that cortisol elicits GS mRNA accumulation by stimulating transcription of the GS gene and that this stimulation requires cell contacts: in dissociated and separated retina cells GS gene transcription was not induced; when the separated cells were reassembled into multicellular aggregates, restoring cell contacts, accumulation of GS mRNA was again inducible. In cells dissociated from retina tissue that had been preinduced with cortisol, GS gene transcription rapidly declined, despite continued hormone availability. In the separated cells transcription of the histone H3.3 gene and accumulation of carbonic anhydrase II mRNA were unaffected; therefore, cell separation selectively precluded induction of the GS gene. These findings provide direct evidence for the regulatory role of cell contacts in hormonal control of gene transcription. Images PMID:2901094

  7. Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

    A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low. Images PMID:2476665

  8. Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

    PubMed Central

    Vardimon, L; Fox, L E; Cohen-Kupiec, R; Degenstein, L; Moscona, A A

    1991-01-01

    Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype. Images PMID:1681425

  9. Glutamine Synthetase Induction in Embryonic Neural Retina: Immunochemical Identification of Polysomes Involved in Enzyme Synthesis

    PubMed Central

    Sarkar, P. K.; Moscona, A. A.

    1973-01-01

    Glutamine synthetase (EC 6.3.1.2) in embryonic neural retina in culture is rapidly induced by hydrocortisone. Retina polysomes involved in translation of this enzyme were precipitated with a high degree of specificity by the gammaglobulin isolated from antiserum against the enzyme (anti-enzyme gammaglobulin). Using immunoprecipitation procedures, we determined that the amount of polysome-bound nascent enzyme was maximal in polysomes comprising 9-14 ribosomes and was about 3-fold higher in the induced than in the noninduced retina. Within this size group of polysomes, those comprising 11-13 ribosomes showed consistently greater binding of anti-enzyme [125I]gammaglobulin than of normal [125I]-gammaglobulin. This size of polysomes corresponds to that calculated for a monocistronic messenger RNA for the subunit of this enzyme, which has a molecular weight of 42,000. The application of immunochemical techniques to identification of templates for synthesis of an enzyme in embryonic cells that constitutes less than 1% of the total cellular proteins indicates the usefulness of this method for detailed studies on regulation of other quantitatively minor products significant in cell differentiation. PMID:4146379

  10. Induction of Glutamine Synthetase Activity in Nonnodulated Roots of Glycine max, Phaseolus vulgaris, and Pisum sativum1

    PubMed Central

    Hoelzle, Inger; Finer, John J.; McMullen, Michael D.; Streeter, John G.

    1992-01-01

    Nitrate or ammonium fertilization significantly increased glutamine synthetase (GS) activity in nonnodulated roots of French bean (Phaseolus vulgaris), soybean (Glycine max), and pea (Pisum sativum). Western analysis revealed substantial GS antibody-positive protein in root extracts that had minimal GS activity, indicating that an inactive form of GS may be present in nonfertilized plants. Images Figure 1 PMID:16652993

  11. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity.

    PubMed Central

    Miller, F W; Waite, K A; Biswas, T; Plotz, P H

    1990-01-01

    Patients with systemic autoimmune diseases make specific autoantibodies that are directed against self structures. According to one view, these autoantibodies arise as a result of an immune response to foreign antigens such as infectious agents that share, by molecular mimicry, common structures with host proteins. An alternative view is that the target autoantigen itself initiates, selects, and sustains autoantibody synthesis. We show here that anti-Jo-1 autoantibodies directed against histidyl-tRNA synthetase in the human autoimmune muscle disease polymyositis undergo, in addition to spectrotype broadening and class switching, the sine qua non of an immune response to the target antigen--affinity maturation to that antigen. We demonstrate further that these autoantibodies, unlike anti-synthetase antibodies induced in mice immunized with heterologous antigen, bind only nonlinear epitopes on the native human synthetase that remain exposed when the enzyme is complexed to tRNA(His). These data suggest that the native target autoantigen itself has played a direct role in selecting and sustaining the autoantibody response and sharply restrict the time and the way in which a molecular mimic might act to provoke autoantibodies. Images PMID:1702223

  12. Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

    PubMed

    Cano, Florencia; Poderoso, Cecilia; Cornejo Maciel, Fabiana; Castilla, Rocío; Maloberti, Paula; Castillo, Fernanda; Neuman, Isabel; Paz, Cristina; Podestá, Ernesto J

    2006-06-01

    The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

  13. CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep.

    PubMed

    Nichani, Anil K; Kaushik, Radhey S; Mena, Angelo; Popowych, Yurij; Dent, Donna; Townsend, Hugh G G; Mutwiri, George; Hecker, Rolf; Babiuk, Lorne A; Griebel, Philip J

    2004-01-01

    Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.

  14. Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

    PubMed

    Manca, Antonella; Sini, Maria Cristina; Izzo, Francesco; Ascierto, Paolo A; Tatangelo, Fabiana; Botti, Gerardo; Gentilcore, Giusy; Capone, Marilena; Mozzillo, Nicola; Rozzo, Carla; Cossu, Antonio; Tanda, Francesco; Palmieri, Giuseppe

    2011-06-01

    Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

  15. Hepatocyte-specific interplay of transcription factors at the far-upstream enhancer of the carbamoylphosphate synthetase gene upon glucocorticoid induction.

    PubMed

    Hoogenkamp, Maarten; Gaemers, Ingrid C; Schoneveld, Onard J L M; Das, Atze T; Grange, Thierry; Lamers, Wouter H

    2007-01-01

    Carbamoylphosphate synthetase-I is the flux-determining enzyme of the ornithine cycle, and neutralizes toxic ammonia by converting it to urea. An 80 bp glucocorticoid response unit located 6.3 kb upstream of the transcription start site mediates hormone responsiveness and liver-specific expression of carbamoylphosphate synthetase-I. The glucocorticoid response unit consists of response elements for the glucocorticoid receptor, forkhead box A, CCAAT/enhancer-binding protein, and an unidentified protein. With only four transcription factor response elements, the carbamoylphosphate synthetase-I glucocorticoid response unit is a relatively simple unit. The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined by comparing a carbamoylphosphate synthetase-I-expressing hepatoma cell line with a carbamoylphosphate synthetase-I-negative fibroblast cell line. DNaseI hypersensitivity assays revealed an open chromatin configuration of the carbamoylphosphate synthetase-I enhancer in hepatoma cells only. In vivo footprinting assays showed that the accessory transcription factors of the glucocorticoid response unit bound to their response elements in carbamoylphosphate synthetase-I-positive cells, irrespective of whether carbamoylphosphate synthetase-I expression was induced with hormones. In contrast, the binding of glucocorticoid receptor to the carbamoylphosphate synthetase-I glucocorticoid response unit was dependent on treatment of the cells with glucocorticoids. Only forkhead box A was exclusively present in hepatoma cells, and therefore appears to be an important determinant of the observed tissue specificity of carbamoylphosphate synthetase-I expression. As the glucocorticoid receptor is the only DNA-binding protein specifically recruited to the glucocorticoid response unit upon stimulation by glucocorticoids, it is likely to be directly responsible for the transcriptional activation mediated by the

  16. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

    1986-01-01

    The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

  17. The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation.

    PubMed

    Shan, Jixiu; Zhang, Fan; Sharkey, Jason; Tang, Tiffany A; Örd, Tönis; Kilberg, Michael S

    2016-11-16

    The response to amino acid (AA) limitation of the entire aminoacyl-tRNA synthetase (ARS) gene family revealed that 16/20 of the genes encoding cytoplasmic-localized enzymes are transcriptionally induced by activating transcription factor 4 (Atf4) via C/ebp-Atf-Response-Element (CARE) enhancers. In contrast, only 4/19 of the genes encoding mitochondrial-localized ARSs were weakly induced. Most of the activated genes have a functional CARE near the transcription start site (TSS), but for others the CARE is downstream. Regardless of the location of CARE enhancer, for all ARS genes there was constitutive association of RNA polymerase II (Pol II) and the general transcription machinery near the TSS. However, for those genes with a downstream CARE, Atf4, C/ebp-homology protein (Chop), Pol II and TATA-binding protein exhibited enhanced recruitment to the CARE during AA limitation. Increased Atf4 binding regulated the association of elongation factors at both the promoter and the enhancer regions, and inhibition of cyclin-dependent kinase 9 (CDK9), that regulates these elongation factors, blocked induction of the AA-responsive ARS genes. Protein pull-down assays indicated that Atf4 directly interacts with CDK9 and its associated protein cyclin T1. The results demonstrate that AA availability modulates the ARS gene family through modulation of transcription elongation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation

    PubMed Central

    Shan, Jixiu; Zhang, Fan; Sharkey, Jason; Tang, Tiffany A.; Örd, Tönis; Kilberg, Michael S.

    2016-01-01

    The response to amino acid (AA) limitation of the entire aminoacyl-tRNA synthetase (ARS) gene family revealed that 16/20 of the genes encoding cytoplasmic-localized enzymes are transcriptionally induced by activating transcription factor 4 (Atf4) via C/ebp-Atf-Response-Element (CARE) enhancers. In contrast, only 4/19 of the genes encoding mitochondrial-localized ARSs were weakly induced. Most of the activated genes have a functional CARE near the transcription start site (TSS), but for others the CARE is downstream. Regardless of the location of CARE enhancer, for all ARS genes there was constitutive association of RNA polymerase II (Pol II) and the general transcription machinery near the TSS. However, for those genes with a downstream CARE, Atf4, C/ebp-homology protein (Chop), Pol II and TATA-binding protein exhibited enhanced recruitment to the CARE during AA limitation. Increased Atf4 binding regulated the association of elongation factors at both the promoter and the enhancer regions, and inhibition of cyclin-dependent kinase 9 (CDK9), that regulates these elongation factors, blocked induction of the AA-responsive ARS genes. Protein pull-down assays indicated that Atf4 directly interacts with CDK9 and its associated protein cyclin T1. The results demonstrate that AA availability modulates the ARS gene family through modulation of transcription elongation. PMID:27471030

  19. [Anti-synthetase syndrome].

    PubMed

    Novak, Srdan

    2012-01-01

    Antysynthetase syndrome is considered as a group ofidiopathic inflammatory myositis with charcteristic serologic hallmark--antibodies which recognise the aminoacyl-tRNA synthetasses (ARS). Clinical picture of those patients contains myositis and/or intersticial lung disease (ILD) and/or arthritis and/or fever and/or Raynaud phenomenon and sometimes characteristic look of mechanic's hands. Myositis can be overt, sometimes even absent, while IBP is major cause of morbidity and determines the outcome of the disease. Untill now eight different any-synthetase autoantibodies are recognised, and most frequent are findings of anti-histidyl-tRNa synthetase antibodies. Patients with other ARS autoantibodies usually have severe ILD. Drug of choice are steroids in dosage of 1 mg/kg with immunosupresive agent (azatioprin or methotrexate) while in severe IBP cyclophosphamide is needed. Recently succsesful treatment with rituximab in combination with cyclophosphamide is reported.

  20. Viral Phosphodiesterases That Antagonize Double-Stranded RNA Signaling to RNase L by Degrading 2-5A

    PubMed Central

    2014-01-01

    The host interferon (IFN) antiviral response involves a myriad of diverse biochemical pathways that disrupt virus replication cycles at many different levels. As a result, viruses have acquired and evolved genes that antagonize the host antiviral proteins. IFNs inhibit viral infections in part through the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L pathway. OAS proteins are pathogen recognition receptors that exist at different basal levels in different cell types and that are IFN inducible. Upon activation by the pathogen-associated molecular pattern viral double-stranded RNA, certain OAS proteins synthesize 2-5A from ATP. 2-5A binds to the antiviral enzyme RNase L causing its dimerization and activation. Recently, disparate RNA viruses, group 2a betacoronaviruses, and group A rotaviruses, have been shown to produce proteins with 2′,5′-phosphodiesterase (PDE) activities that eliminate 2-5A thereby evading the antiviral activity of the OAS/RNase L pathway. These viral proteins are members of the eukaryotic-viral LigT-like group of 2H phosphoesterases, so named for the presence of 2 conserved catalytic histidine residues. Here, we will review the biochemistry, biology, and implications of viral and cellular 2′,5′-PDEs that degrade 2-5A. In addition, we discuss alternative viral and cellular strategies for limiting the activity of OAS/RNase L. PMID:24905202

  1. Full activation of RNaseL in animal cells requires binding of 2-5A within ankyrin repeats 6 to 9 of this interferon-inducible enzyme.

    PubMed

    Díaz-Guerra, M; Rivas, C; Esteban, M

    1999-02-01

    To define protein domains important for activation of the interferon (IFN)-induced enzyme 2-5A-dependent RNaseL, we have generated vaccinia virus (VV) recombinants able to express in cultured cells truncated forms of this protein and compared their biologic activities with those producing the wild-type enzyme, with and without coexpression of 2-5A synthetase. Our results show that full activation of RNaseL requires binding of 2-5A oligonucleotides within amino acid positions 212-339, corresponding to ankyrin repeats 6 to 9. The protein kinase and ribonuclease domains of RNaseL, amino acids 340-741, are sufficient for a constitutively active enzyme that is unresponsive to excess 2-5A. These results demonstrate in vivo the importance of the ankyrin domains in the biologic function of RNaseL. We suggest that ankyrin repeats act as key modulators of RNaseL activity.

  2. In vitro effects of metal pollution on Mediterranean sponges: species-specific inhibition of 2',5'-oligoadenylate synthetase.

    PubMed

    Saby, Emilie; Justesen, Just; Kelve, Merike; Uriz, Maria J

    2009-09-14

    Heavy metals are among the main pollutants of the Mediterranean coastal waters where they can harm sublittoral biota. Filter-feeder, long-living invertebrates that remain fixed to the rocky bottom, such as sponges, are good targets to metal contamination studies since they may be exposed to potential low levels of contamination for years. Several molecular and biochemical mechanisms are developed by sponges to counteract the effects of noxious metals. As a result, some of the normal cell functions can be altered. Here we show that the main heavy metals that can be found in marine sublittoral waters (i.e. copper, iron, zinc and manganese) may alter the immune system of sponges by inhibiting the activity of the sponge 2',5'-oligoadenylate synthetase (2-5A synthetase), which is an enzyme involved in the immune system of vertebrates. We selected the widespread Mediterranean sponges Geodia cydonium, Crella elegans and Chondrosia reniformis for the study. They exerted a high 2-5A synthetase activity and gave a unique profile of 2',5'-oligoadenylate product production. Several metals alter the 2-5A synthetase activity differently, in a species-specific manner. 2-5A synthetases from G. cydonium and C. elegans were inhibited by all the metal ions assayed. However, in C. reniformis, 2-5A synthetase was either activated or inhibited by the same ions depending on their final concentrations. Like in humans, metal contamination may have an effect on the OAS activity and thus it might alter the sponge immune system. However, since the effects are species-specific, 2-5A synthetase cannot be used as general biomarker of metal pollutions.

  3. In vitro and in vivo effect of interleukin-2 on the 2',5'-oligoadenylate synthetase activity of peripheral mononuclear blood cells

    SciTech Connect

    Handgretinger, R.; Bruchelt, G.; Kimmig, A.; Lang, P.; Daurer, B.; Dopfer, R.; Treuner, J.; Niethammer, D. )

    1990-02-01

    The in vitro and in vivo influence of interleukin-2 (IL-2) on 2',5'-oligoadenylate (2-5A) synthetase activity and natural killer (NK) activity of peripheral mononuclear blood cells (PBMCs) was investigated. Incubation of PBMCs in vitro with IL-2 resulted in a considerable secretion of interferon-gamma (IFN-gamma) and in a significant elevation of 2-5A synthetase activity, as well as NK activity. Neutralizing monoclonal anti-IFN-gamma antibodies inhibited the elevation of 2-5A synthetase activity, but not the IL-2-induced augmentation of NK activity. Additionally, 2-5A synthetase and NK activity of PBMCs was measured in a child with neuroblastoma that was treated with recombinant IL-2 by continuous intravenous application. During the treatment, NK activity against the NK-sensitive cell line K 562 and against autologous tumor cells was not augmented. However, a significant increase of 2-5A synthetase activity in PBMCs was observed during IL-2 treatment, whereas there was no detectable serum level of IFN-gamma. We conclude that measuring 2-5A synthetase activity in patients treated with IL-2 may be helpful in monitoring the immunomodulatory effects of IL-2 on immune effector cells.

  4. Interruption of myogenesis by transforming growth factor beta 1 or EGTA inhibits expression and activity of the myogenic-associated (2'-5') oligoadenylate synthetase and PKR.

    PubMed

    Salzberg, S; Mandelboim, M; Zalcberg, M; Shainberg, A

    1995-07-01

    Interferon-induced proteins have been previously implicated in the regulation of cell growth. In an attempt to provide evidence for the involvement of these proteins in differentiation, the effect of transforming growth factor beta 1 (TGF-beta) and EGTA on the expression and activity of (2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA activated protein kinase (PKR) during myogenesis of rat primary skeletal muscle cultures or the myogenic cell line L8 was studied. Both TGF-beta and EGTA inhibited the fusion of myoblasts and reduced significantly the level of the muscle-specific proteins, acetylcholine receptors, and creatine kinase activity in rat primary muscle cultures. Likewise, TGF-beta exhibited a similar inhibitory effect on the fusion of L8 cells and the level of creatine kinase activity in these cells. The kinetics of 2-5A synthetase activity in both types of cells during differentiation was then established. In both types, a transient increase in activity was observed followed by a decrease thereafter. However, while the peak activity in primary muscle cultures appeared after 24 h in culture, it was observed only on the third day in L8 cells grown in differentiation medium (DM). Treatment of primary cultures with either TGF-beta or EGTA reduced the amount of 1.7-kb 2-5A synthetase-specific RNA transcripts and decreased significantly the level of 2-5A synthetase activity compared to that in untreated cultures. Western blot analysis of 2-5A synthetase proteins in untreated primary muscle cultures showed that the major species synthesized in these cells was the 43-kDa isoform of the enzyme. However, the 71-kDa isoform was clearly visible after 72 h in culture. Both TGF-beta and EGTA abrogated the appearance of all forms of 2-5A synthetase. Similarly, in L8 cells grown in DM, TGF-beta down-regulated the expression of 2-5A synthetase and reduced the level of enzymatic activity. Western blot analysis revealed the presence of the 71-k

  5. Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA

    PubMed Central

    Player, Mark R.; Barnard, Dale L.; Torrence, Paul F.

    1998-01-01

    The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication. PMID:9671772

  6. Genetics Home Reference: glutathione synthetase deficiency

    MedlinePlus

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions glutathione synthetase deficiency glutathione synthetase ...

  7. The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield1[OPEN

    PubMed Central

    He, Xue; Qu, Baoyuan; Li, Wenjing; Zhao, Xueqiang; Teng, Wan; Ma, Wenying; Ren, Yongzhe; Li, Bin; Li, Zhensheng; Tong, Yiping

    2015-01-01

    Nitrate is a major nitrogen resource for cereal crops; thus, understanding nitrate signaling in cereal crops is valuable for engineering crops with improved nitrogen use efficiency. Although several regulators have been identified in nitrate sensing and signaling in Arabidopsis (Arabidopsis thaliana), the equivalent information in cereals is missing. Here, we isolated a nitrate-inducible and cereal-specific NAM, ATAF, and CUC (NAC) transcription factor, TaNAC2-5A, from wheat (Triticum aestivum). A chromatin immunoprecipitation assay showed that TaNAC2-5A could directly bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthetase. Overexpression of TaNAC2-5A in wheat enhanced root growth and nitrate influx rate and, hence, increased the root’s ability to acquire nitrogen. Furthermore, we found that TaNAC2-5A-overexpressing transgenic wheat lines had higher grain yield and higher nitrogen accumulation in aerial parts and allocated more nitrogen in grains in a field experiment. These results suggest that TaNAC2-5A is involved in nitrate signaling and show that it is an exciting gene resource for breeding crops with more efficient use of fertilizer. PMID:26371233

  8. Hepatocytes explanted in the spleen preferentially express carbamoylphosphate synthetase rather than glutamine synthetase.

    PubMed

    Lamers, W H; Been, W; Charles, R; Moorman, A F

    1990-10-01

    Urea cycle enzymes and glutamine synthetase are essential for NH3 detoxification and systemic pH homeostasis in mammals. Carbamoylphosphate synthetase, the first and flux-determining enzyme of the cycle, is found only in a large periportal compartment, and glutamine synthetase is found only in a small, complementary pericentral compartment. Because it is not possible to manipulate experimentally the intrahepatic distribution of carbamoylphosphate synthetase and glutamine synthetase, we looked for conditions in which explanted hepatocytes would exhibit either the carbamoylphosphate synthetase phenotype or glutamine synthetase phenotype. In the spleen hepatocytes either settle as individual cells or in small agglomerates. The dispersed cells only express the carbamoylphosphate synthetase phenotype. Within the agglomerates, sinusoids that drain on venules develop. Hepatocytes surrounding the venules stain only weakly for carbamoylphosphate synthetase but are strongly positive for glutamine synthetase. These observations were made for explanted embryonic hepatocytes (no prior expression of either carbamoylphosphate synthetase or glutamine synthetase), neonatal hepatocytes (compartments of gene expression not yet established) and adult periportal and pericentral hepatocytes.

  9. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    MedlinePlus

    ... Health Conditions carbamoyl phosphate synthetase I deficiency carbamoyl phosphate synthetase I deficiency Printable PDF Open All Close ... to view the expand/collapse boxes. Description Carbamoyl phosphate synthetase I deficiency is an inherited disorder that ...

  10. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    SciTech Connect

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  11. Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation.

    PubMed

    Chang, Yu-Fang; Martin, Shelley S; Baldwin, Enoch P; Carman, George M

    2007-06-15

    Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser(462) and Thr(455) were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.

  12. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION*

    PubMed Central

    Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

    2007-01-01

    Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time-and dose-dependent, and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1R398A-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Δ ura8Δ mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser462 and Thr455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser462 and Thr455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibits a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367–5377). These data indicated that protein kinase C phosphorylation at Ser462 stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr455 inhibits activity. PMID:17463002

  13. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

    PubMed

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just; Kelve, Merike; Uriz, Maria Jesus

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

  14. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  15. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  16. Genetics Home Reference: holocarboxylase synthetase deficiency

    MedlinePlus

    ... use of biotin, a B vitamin found in foods such as liver, egg yolks, and milk. Holocarboxylase synthetase attaches biotin to certain enzymes that are essential for the normal production and breakdown of proteins, fats, and carbohydrates in ...

  17. Cell-Type-Specific Effects of RNase L on Viral Induction of Beta Interferon

    PubMed Central

    Banerjee, Shuvojit; Chakrabarti, Arindam; Jha, Babal Kant; Weiss, Susan R.; Silverman, Robert H.

    2014-01-01

    ABSTRACT The interferon (IFN)-inducible antiviral state is mediated in part by the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections. PMID:24570368

  18. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

    PubMed

    Pérez-Delgado, Carmen M; García-Calderón, Margarita; Márquez, Antonio J; Betti, Marco

    2015-01-01

    It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.

  19. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

    PubMed Central

    Pérez-Delgado, Carmen M.; García-Calderón, Margarita; Márquez, Antonio J.; Betti, Marco

    2015-01-01

    It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4+ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity. PMID:26091523

  20. Injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2'-5' oligoadenylate synthetase in peritoneal macrophages.

    PubMed Central

    Gresser, I; Vignaux, F; Belardelli, F; Tovey, M G; Maunoury, M T

    1985-01-01

    Injection of conventional or axenic weanling mice with potent sheep or goat antibody to mouse interferon alpha/beta resulted in a decrease in the basal level of 2-5A synthetase in resting peritoneal macrophages and rendered these cells permissive for vesicular stomatitis virus. There was a good inverse correlation between the level of 2-5A synthetase in peritoneal macrophages and the permissivity of these cells for vesicular stomatitis virus. The peritoneal macrophages of 1- and 2-week-old mice had low levels of 2-5A synthetase and were permissive for vesicular stomatitis virus, whereas at 3 weeks (and after) there was a marked increase in the level of 2-5A synthetase in peritoneal macrophages, and these cells were no longer permissive for vesicular stomatitis virus. We suggest that low levels of interferon alpha or beta or both are produced in normal mice, and that this interferon contributes to host defense by inducing and maintaining an antiviral state in some cells. PMID:2981340

  1. Regulation of 2', 5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor

    SciTech Connect

    Garcia-Blanco, M.A. ); Lengyel, P. . Dept. of Molecular Biophysics and Biochemistry); Morrison, E.; BrownLee, C.; Stiles, C.D. ); Williams, B.R.G. )

    1989-03-01

    In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2', 5-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta inteferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2', 5-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2'-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.

  2. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    PubMed

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  3. Fatty Acid Synthetase of Saccharomyces cerevisiae

    PubMed Central

    Klein, Harold P.; Volkmann, Carol M.; Chao, Fu-Chuan

    1967-01-01

    A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein. Images PMID:6025308

  4. A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

    PubMed

    Ibba, M; Morgan, S; Curnow, A W; Pridmore, D R; Vothknecht, U C; Gardner, W; Lin, W; Woese, C R; Söll, D

    1997-11-07

    The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

  5. Regulation of Acetyl Coenzyme A Synthetase in Escherichia coli

    PubMed Central

    Kumari, Suman; Beatty, Christine M.; Browning, Douglas F.; Busby, Stephen J. W.; Simel, Erica J.; Hovel-Miner, Galadriel; Wolfe, Alan J.

    2000-01-01

    Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways. PMID:10894724

  6. Conformational analysis of brominated pA2'-5'A2'-5'A analogs. An NMR and model-building study.

    PubMed

    van den Hoogen, Y T; Hilgersom, C M; Brozda, D; Lesiak, K; Torrence, P F; Altona, C

    1989-07-01

    NMR and model-building studies were carried out on pA2'-5'A2'-5'A and analogs in which one or more of the A residues were replaced by 8-bromoadenosine. Chemical shifts, coupling constants and NOE data were used to obtain structural information. The N/S equilibrium constant of the ribose rings as well as the phase angles and puckering amplitudes were determined from the experimental coupling constants with the aid of an improved version of the PSEUROT program. Chemical shifts in combination with NOE data were used to monitor base-base interactions and the orientation of the bases (syn or anti). The combined data suggest that different types of stacking interactions are present in the various compounds. Bromination of the first or second residue in the trimers results in a preference for N-type sugar and syn orientation of the base in these residues. When A(3) is brominated, an S-type sugar conformation together with a syn orientation of the base is favoured at the 2' terminus. Energy-minimized models of the different stacking interactions are presented which fit the present collection of data. The possible correlation between biological activity of these compounds and their conformation is briefly discussed.

  7. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  8. Peptide Synthetase Gene in Trichoderma virens

    PubMed Central

    Wilhite, S. E.; Lumsden, R. D.; Straney, D. C.

    2001-01-01

    Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used. PMID:11679326

  9. Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones

    PubMed Central

    1979-01-01

    Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L- thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones. PMID:117014

  10. Urease of Klebsiella aerogenes: control of its synthesis by glutamine synthetase.

    PubMed Central

    Friedrich, B; Magasanik, B

    1977-01-01

    Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate. PMID:18438

  11. Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.

    PubMed Central

    Smith, K.; Nadeau, K.; Bradley, M.; Walsh, C.; Fairlamb, A. H.

    1992-01-01

    Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo. PMID:1304372

  12. Cell-type-specific effects of RNase L on viral induction of beta interferon.

    PubMed

    Banerjee, Shuvojit; Chakrabarti, Arindam; Jha, Babal Kant; Weiss, Susan R; Silverman, Robert H

    2014-02-25

    The interferon (IFN)-inducible antiviral state is mediated in part by the 2',5'-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections. IMPORTANCE Type I interferons (IFNs) such as IFN-β are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-β production in mouse

  13. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

    PubMed Central

    Dworeck, Tamara; Zimmermann, Martin

    2012-01-01

    Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (km 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (km 0,07) and lowered the affinity for Gly (km 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (km 0,23) as a substrate, while affinity to Gly was preserved (km 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event. PMID:23091597

  14. In vivo modification of Azotobacter chroococcum glutamine synthetase.

    PubMed

    Muñoz-Centeno, M C; Cejudo, F J; Paneque, A

    1994-03-15

    A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.

  15. In vivo modification of Azotobacter chroococcum glutamine synthetase.

    PubMed Central

    Muñoz-Centeno, M C; Cejudo, F J; Paneque, A

    1994-01-01

    A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7908189

  16. Retinal Vasculitis in Anti-Synthetase Syndrome.

    PubMed

    Donovan, Christopher P; Pecen, Paula E; Baynes, Kimberly; Ehlers, Justis P; Srivastava, Sunil K

    2016-09-01

    A 31-year-old woman with a history of anti-synthetase syndrome-related myositis and interstitial lung disease presented with acute-onset blurry vision and rash on her hands and feet. Visual acuity was hand motion in her right eye and 20/40 in her left eye. Dilated fundus exam showed extensive retinal vasculitis, diffuse intraretinal hemorrhages, and subretinal fluid. Optical coherence tomography revealed significant macular thickening, and fluorescein angiography revealed vascular leakage with peripheral nonperfusion. Aggressive systemic immunosuppression was initiated, with gradual resolution of her disease during 8 months of follow-up. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:874-879.].

  17. Characterization of Cereulide Synthetase, a Toxin-Producing Macromolecular Machine

    PubMed Central

    Alonzo, Diego A.; Magarvey, Nathan A.; Schmeing, T. Martin

    2015-01-01

    Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride. PMID:26042597

  18. Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation

    DTIC Science & Technology

    1991-06-26

    extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM... wheat germ extract . A cDNA clone coding for 2-5A-depRNase was isolated (B. Hassel, A. Zhou, and R.H. Silverman, unpublished), in vitro transcribed...and the corresponding mRNA was in vitro translated in cell free extracts of wheat germ . In vitro transcription. Incubations were set up in 0.5 ml

  19. FRET monitoring of a nonribosomal peptide synthetase.

    PubMed

    Alfermann, Jonas; Sun, Xun; Mayerthaler, Florian; Morrell, Thomas E; Dehling, Eva; Volkmann, Gerrit; Komatsuzaki, Tamiki; Yang, Haw; Mootz, Henning D

    2017-09-01

    Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

  20. Aminoacyl-tRNA synthetases database.

    PubMed

    Szymanski, M; Deniziak, M A; Barciszewski, J

    2001-01-01

    Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases' evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and Söll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202-236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobases.ibch.poznan.pl/aars/.

  1. The microsomal dicarboxylyl-CoA synthetase.

    PubMed Central

    Vamecq, J; de Hoffmann, E; Van Hoof, F

    1985-01-01

    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo. PMID:4062873

  2. Glutamine Synthetase: Role in Neurological Disorders.

    PubMed

    Jayakumar, Arumugam R; Norenberg, Michael D

    2016-01-01

    Glutamine synthetase (GS) is an ATP-dependent enzyme found in most species that synthesizes glutamine from glutamate and ammonia. In brain, GS is exclusively located in astrocytes where it serves to maintain the glutamate-glutamine cycle, as well as nitrogen metabolism. Changes in the activity of GS, as well as its gene expression, along with excitotoxicity, have been identified in a number of neurological conditions. The literature describing alterations in the activation and gene expression of GS, as well as its involvement in different neurological disorders, however, is incomplete. This review summarizes changes in GS gene expression/activity and its potential contribution to the pathogenesis of several neurological disorders, including hepatic encephalopathy, ischemia, epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and astroglial neoplasms. This review also explores the possibility of targeting GS in the therapy of these conditions.

  3. Mechanistic issues in asparagine synthetase catalysis.

    PubMed

    Richards, N G; Schuster, S M

    1998-01-01

    The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred.

  4. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  5. Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia coli

    PubMed Central

    Newton, W. Austin; Snell, Esmond E.

    1965-01-01

    Newton, W. Austin (University of California, Berkeley), and Esmond E. Snell. Formation and interrelationships of tryptophanase and tryptophan synthetases in Escherichia coli. J. Bacteriol. 89:355–364. 1965.—In addition to the classical tryptophan-repressible tryptophan synthetase (TSase-tr), tryptophan auxotrophs of Escherichia coli contain another distinct tryptophan synthetase (TSase-ti) which is induced by tryptophan and is identical with tryptophanase (TPase). Escherichia coli B (wild type) forms only TSase-tr when the growth medium lacks tryptophan. When tryptophan is supplied, parallel induction of TPase and TSase-ti occurs while TSase-tr is repressed. Antiserum prepared against purified TPase neutralized TPase and TSase-ti equally, but not TSase-tr. TPase-negative strains of E. coli do not form TSase-ti. Unlike TSase-tr, TSase-ti is not readily detected by whole-cell assays. In the tryptophan auxotroph, E. coli B/1t7, a direct correlation exists between the effectiveness of 4-, 5-, and 6-methyl-tryptophan in inducing TPase and in promoting growth in the presence of indole. In a mutant of this organism, E. coli B/1t7-A, which is constitutive for TPase, 5-methyl-tryptophan and other substrates of TPase increased the rate of growth on limiting indole, a result ascribed to their ability to inhibit degradation of tryptophan and to supply the 3-carbon side chain for synthesis of tryptophan by TPase. This organism produced maximal amounts of TPase when inocula from log-phase cells grown in tryptophan-supplemented minimal medium were allowed to undergo two cell generations in an enriched broth medium. PMID:14255701

  6. Crystal structure of human annexin I at 2.5 A resolution.

    PubMed Central

    Weng, X.; Luecke, H.; Song, I. S.; Kang, D. S.; Kim, S. H.; Huber, R.

    1993-01-01

    cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I). PMID:8453382

  7. Purification and properties of glutamine synthetase from spinach leaves.

    PubMed

    Ericson, M C

    1985-12-01

    The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem ;reactive blue A-agarose' and ;reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K(m) is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K(i) values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase.

  8. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

  9. Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase.

    PubMed Central

    Sanni, A; Walter, P; Boulanger, Y; Ebel, J P; Fasiolo, F

    1991-01-01

    Phenylalanyl-tRNA synthetases [L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20] from Escherichia coli, yeast cytoplasm, and mammalian cytoplasm have an unusual conserved alpha 2 beta 2 quaternary structure that is shared by only one other aminoacyl-tRNA synthetase. Both subunits are required for activity. We show here that a single mitochondrial polypeptide from Saccharomyces cerevisiae is an active phenylalanyl-tRNA synthetase. This protein (the MSF1 gene product) is active as a monomer. It has all three characteristic sequence motifs of the class II aminoacyl-tRNA synthetases, and its activity may result from the recruitment of additional sequences into an alpha-subunit-like structure. Images PMID:1924298

  10. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  11. Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

    PubMed

    Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A G; Santamaría-Gómez, Javier; Patterson, Carl J; Foster, Andrew W; Bru-Martínez, Roque; Robinson, Nigel J; Luque, Ignacio

    2015-11-16

    Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

  12. Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress

    PubMed Central

    Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A. G.; Santamaría-Gómez, Javier; Patterson, Carl J.; Foster, Andrew W.; Bru-Martínez, Roque; Robinson, Nigel J.; Luque, Ignacio

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNAThr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNAThr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs. PMID:26464444

  13. Glutamine synthetase induced spinal seizures in rats.

    PubMed

    Shin, Dong Won; Yoon, Young Sul; Matsumoto, Masato; Huang, Wencheng; Ceraulo, Phil; Young, Wise

    2003-02-01

    Glutamine synthetase (GS) is a key enzyme in the regulation of glutamate neurotransmission in the central nervous system. It is responsible for converting glutamate to glutamine, consuming one ATP and NH3 in the process. Glutamate is neurotoxic when it accumulates in extracellular fluids. We investigated the effects of GS in both a spinal cord injury (SCI) model and normal rats. 0.1-ml of low (2- micro M) and high (55- micro M) concentrations of GS were applied, intrathecally, to the spinal cord of rats under pentobarbital anesthesia. Immediately after an intrathecal injection into the L1-L3 space, the rats developed convulsive movements. These movements initially consisted of myoclonic twitches of the paravertebral muscles close to the injection site, repeated tonic and clonic contractions and extensions of the hind limbs (hind limb seizures) that spread to the fore limbs, and finally rotational axial movements of the body. An EMG of the paravertebral muscles, fore and hind limbs, showed the extent of the muscle activities. GS (2- micro M) caused spinal seizures in the rats after the SCI, and GS (6- micro M) produced seizures in the uninjured anesthetized rats. Denatured GS (70 degrees C, 1 hour) also produced spinal seizures, although higher concentrations were required. We suggest that GS may be directly blocking the release of GABA, or the receptors, in the spinal cord.

  14. Chemical modification of E. coli glutamine synthetase

    SciTech Connect

    DiIanni, C.L.; Colanduoni, J.A.; Collins, R.; Villafranca, J.J.

    1986-05-01

    Thiourea trioxide partially inactivates E. coli glutamine synthetase (GS) (approx.25%) by reacting only with lysine residues, producing homoarginine. Thiourea dioxide totally inactivates GS by reacting with both lysine and histidine residues. The K/sub m/ values for thiourea trioxide modified enzyme are 0.21 mM for ATP and 10 mM for glutamate which are about threefold higher than for native GS. Using (/sup 14/C) thiourea trioxide, 2.3 +/- 0.2 moles of reagent were incorporated per monomer. The same number of homoarginine residues were found by amino acid analysis. Modification of GS with hydroxylamine results in total inactivation resulting from reaction with histidine. Fluorescence titrations indicate that substrate binding to the modified enzyme is weaker than to the native enzyme. EPR spectra of bound Mn/sup 2 +/ indicate that metal ion binding is unaffected by hydroxylamine modification. However, metal ion binding is weaker to the modified enzyme. Protection from hydroxylamine inactivation is observed with ATP + Glutamate, AMPPNP + Glutamate, and MgCl/sub 2/.

  15. Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

    SciTech Connect

    Banerjee, R.V.; Shane, B.; McGuire, J.J.; Coward, J.K.

    1988-12-13

    The transfer of /sup 17/O and/or /sup 18/O from (COOH-/sup 17/O or -/sup 18/O) enriched substrates to inorganic phosphate (P/sub i/) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-/sup 18/O-labeled folate, methotrexate, and dihydropteroate, in addition to (/sup 17/O)-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. P/sub i/ was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for /sup 17/O and /sup 18/O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate /sup 18/O-enriched carboxyl group to P/sub i/ occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of P/sub i/ obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that /sup 18/O transfer had occurred.

  16. Pyrrolysyl-tRNA synthetase, an aminoacyl-tRNA synthetase for genetic code expansion

    PubMed Central

    Crnković, Ana; Suzuki, Tateki; Söll, Dieter; Reynolds, Noah M.

    2016-01-01

    Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS. PMID:28239189

  17. The mammalian 2'-5' oligoadenylate synthetase gene family: evidence for concerted evolution of paralogous Oas1 genes in Rodentia and Artiodactyla.

    PubMed

    Perelygin, Andrey A; Zharkikh, Andrey A; Scherbik, Svetlana V; Brinton, Margo A

    2006-10-01

    Multiple 2'-5' oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2'-5' oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight Oas1 genes, two Oas1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2'-5' oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection.

  18. Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25.

    PubMed

    Perelygin, A A; Lear, T L; Zharkikh, A A; Brinton, M A

    2005-01-01

    Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.

  19. Aminoacyl tRNA synthetases and their connections to disease.

    PubMed

    Park, Sang Gyu; Schimmel, Paul; Kim, Sunghoon

    2008-08-12

    Aminoacylation of transfer RNAs establishes the rules of the genetic code. The reactions are catalyzed by an ancient group of 20 enzymes (one for each amino acid) known as aminoacyl tRNA synthetases (AARSs). Surprisingly, the etiology of specific diseases-including cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions-is connected to specific aminoacyl tRNA synthetases. These connections include heritable mutations in the genes for tRNA synthetases that are causally linked to disease, with both dominant and recessive disease-causing mutations being annotated. Because some disease-causing mutations do not affect aminoacylation activity or apparent enzyme stability, the mutations are believed to affect functions that are distinct from aminoacylation. Examples include enzymes that are secreted as procytokines that, after activation, operate in pathways connected to the immune system or angiogenesis. In addition, within cells, synthetases form multiprotein complexes with each other or with other regulatory factors and in that way control diverse signaling pathways. Although much has been uncovered in recent years, many novel functions, disease connections, and interpathway connections of tRNA synthetases have yet to be worked out.

  20. Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

    PubMed

    Hewagama, A; Guy, H I; Vickrey, J F; Evans, D R

    1999-10-01

    Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.

  1. The glutamine synthetase gene family in Populus

    PubMed Central

    2011-01-01

    Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. Conclusions Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types. PMID:21867507

  2. Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

    NASA Astrophysics Data System (ADS)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

  3. tRNA synthetase: tRNA Aminoacylation and beyond

    PubMed Central

    Pang, Yan Ling Joy; Poruri, Kiranmai; Martinis, Susan A.

    2014-01-01

    The aminoacyl-tRNA synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. Each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of tRNA isoacceptors. These ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their aminoacylation role and impact a wide range of other metabolic pathways and cell signaling processes. The family of aminoacyl-tRNA synthetases have also been suggested as a remarkable scaffold to incorporate new domains that would drive evolution and the emergence of new organisms with more complex function. Because they are essential, the tRNA synthetases have served as pharmaceutical targets for drug and antibiotic development. The recent unfolding of novel important functions for this family of proteins offers new and promising pathways for therapeutic development to treat diverse human diseases. PMID:24706556

  4. Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L. 1

    PubMed Central

    Lara, Miguel; Porta, Helena; Padilla, Jaime; Folch, Jorge; Sánchez, Federico

    1984-01-01

    Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined. The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N2-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides. Images Fig. 1 Fig. 2 Fig. 4 PMID:16663942

  5. Inhibition of Plant Glutamine Synthetases by Substituted Phosphinothricins

    PubMed Central

    Logusch, Eugene W.; Walker, Daniel M.; McDonald, John F.; Franz, John E.

    1991-01-01

    Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS1 and chloroplastic GS2 competitively versusl-glutamate, with Ki values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low Ki values and slow enzyme turnover, as described by reactivation characteristics. Both the GS1 and GS2 isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series. PMID:16668090

  6. Recurrent Isolated Neonatal Hemolytic Anemia: Think About Glutathione Synthetase Deficiency.

    PubMed

    Signolet, Isabelle; Chenouard, Rachel; Oca, Florine; Barth, Magalie; Reynier, Pascal; Denis, Marie-Christine; Simard, Gilles

    2016-09-01

    Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA. Copyright © 2016 by the American Academy of Pediatrics.

  7. Interferon-induced 2'-5' adenylate synthetase in vivo and interferon production in vitro by lymphocytes from systemic lupus erythematosus patients with and without circulating interferon

    SciTech Connect

    Preble, O.T.; Rothko, K.; Klippel, J.H.; Friedman, R.M.; Johnston, M.I.

    1983-06-01

    The interferon (IFN)-induced enzyme 2-5A synthetase was elevated in mononuclear cells from both serum IFN-positive and -negative systemic lupus erythematosus (SLE) patients. This suggests that a much higher percentage of patients than previously thought produce endogenous IFN. These results may partly explain findings that mononuclear cells from SLE patients are deficient in IFN production in vitro in response to certain IFN inducers. Although normal lymphocytes can produce an acid-labile alpha IFN after stimulation with C. parvum in vitro, the reason for endogenous production of this unusual alpha IFN by SLE patients remains unknown.

  8. The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase*

    PubMed Central

    Frago, Susana; Velázquez-Campoy, Adrián; Medina, Milagros

    2009-01-01

    In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg2+. The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg2+ and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced by adenine nucleotide-Mg2+ or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains. PMID:19136717

  9. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    PubMed

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  10. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

    PubMed Central

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

    2014-01-01

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

  11. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling.

    PubMed

    Bernard, Stéphanie M; Habash, Dimah Z

    2009-01-01

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  12. Decreased Red Cell Uroporphyrinogen I Synthetase Activity in Intermittent Acute Porphyria

    PubMed Central

    Strand, L. James; Meyer, Urs A.; Felsher, Bertram F.; Redeker, Allan G.; Marver, Harvey S.

    1972-01-01

    Intermittent acute porphyria has recently been distinguished biochemically from other genetic hepatic porphyrias by the observation of diminished hepatic uroporphyrinogen I synthetase activity and increased δ-aminolevulinic acid synthetase activity. Since deficient uroporphyrinogen I synthetase may be reflected in nonhepatic tissues, we have assayed this enzyme in red cell hemolysates from nonporphyric subjects and from patients with genetic hepatic porphyria. Only patients with intermittent acute porphyria had decreased erythrocyte uroporphyrinogen I synthetase activity which was approximately 50% of normal. The apparent Km of partially purified uroporphyrinogen I synthetase was 6 × 10−6m in both nonporphyrics and patients with intermittent acute porphyria. These data provide further evidence for a primary mutation affecting uroporphyrinogen I synthetase in intermittent acute porphyria. Further-more, results of assay of red cell uroporphyrinogen I synthetase activity in a large family with intermittent acute porphyria suggest that this test may be a reliable indicator of the heterozygous state. PMID:5056653

  13. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    SciTech Connect

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  14. Gravitational induction

    NASA Astrophysics Data System (ADS)

    Bini, Donato; Cherubini, Christian; Chicone, Carmen; Mashhoon, Bahram

    2008-11-01

    We study the linear post-Newtonian approximation to general relativity known as gravitoelectromagnetism (GEM); in particular, we examine the similarities and differences between GEM and electrodynamics. Notwithstanding some significant differences between them, we find that a special nonstationary metric in GEM can be employed to show explicitly that it is possible to introduce gravitational induction within GEM in close analogy with Faraday's law of induction and Lenz's law in electrodynamics. Some of the physical implications of gravitational induction are briefly discussed.

  15. Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity

    PubMed Central

    Huang, Hao; Zeqiraj, Elton; Dong, Beihua; Jha, Babal Kant; Duffy, Nicole; Orlicky, Stephen; Thevakumaran, Neroshan; Talukdar, Manisha; Pillon, Monica C.; Ceccarelli, Derek F.; Wan, Leo; Juang, Yu-Chi; Mao, Daniel Y.L.; Gaughan, Christina; Brinton, Margo A.; Perelygin, Andrey A.; Kourinov, Igor; Guarné, Alba; Silverman, Robert H.; Sicheri, Frank

    2014-01-01

    Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold. PMID:24462203

  16. Inductive reasoning.

    PubMed

    Hayes, Brett K; Heit, Evan; Swendsen, Haruka

    2010-03-01

    Inductive reasoning entails using existing knowledge or observations to make predictions about novel cases. We review recent findings in research on category-based induction as well as theoretical models of these results, including similarity-based models, connectionist networks, an account based on relevance theory, Bayesian models, and other mathematical models. A number of touchstone empirical phenomena that involve taxonomic similarity are described. We also examine phenomena involving more complex background knowledge about premises and conclusions of inductive arguments and the properties referenced. Earlier models are shown to give a good account of similarity-based phenomena but not knowledge-based phenomena. Recent models that aim to account for both similarity-based and knowledge-based phenomena are reviewed and evaluated. Among the most important new directions in induction research are a focus on induction with uncertain premise categories, the modeling of the relationship between inductive and deductive reasoning, and examination of the neural substrates of induction. A common theme in both the well-established and emerging lines of induction research is the need to develop well-articulated and empirically testable formal models of induction. Copyright © 2010 John Wiley & Sons, Ltd. For further resources related to this article, please visit the WIREs website.

  17. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase.

    PubMed

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M; Vega, José M; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress.

  18. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase

    PubMed Central

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M.; Vega, José M.; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J.; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress. PMID:26442073

  19. Crystal structure of the archaeal asparagine synthetase: interrelation with aspartyl-tRNA and asparaginyl-tRNA synthetases.

    PubMed

    Blaise, Mickaël; Fréchin, Mathieu; Oliéric, Vincent; Charron, Christophe; Sauter, Claude; Lorber, Bernard; Roy, Hervé; Kern, Daniel

    2011-09-23

    Asparagine synthetase A (AsnA) catalyzes asparagine synthesis using aspartate, ATP, and ammonia as substrates. Asparagine is formed in two steps: the β-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. Interestingly, this mechanism of amino acid activation resembles that used by aminoacyl-tRNA synthetases, which first activate the α-carboxylate group of the amino acid to form also an aminoacyl-AMP before they transfer the activated amino acid onto the cognate tRNA. In a previous investigation, we have shown that the open reading frame of Pyrococcus abyssi annotated as asparaginyl-tRNA synthetase (AsnRS) 2 is, in fact, an archaeal asparagine synthetase A (AS-AR) that evolved from an ancestral aspartyl-tRNA synthetase (AspRS). We present here the crystal structure of this AS-AR. The fold of this protein is similar to that of bacterial AsnA and resembles the catalytic cores of AspRS and AsnRS. The high-resolution structures of AS-AR associated with its substrates and end-products help to understand the reaction mechanism of asparagine formation and release. A comparison of the catalytic core of AS-AR with those of archaeal AspRS and AsnRS and with that of bacterial AsnA reveals a strong conservation. This study uncovers how the active site of the ancestral AspRS rearranged throughout evolution to transform an enzyme activating the α-carboxylate group into an enzyme that is able to activate the β-carboxylate group of aspartate, which can react with ammonia instead of tRNA. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori.

    PubMed

    Hu, Jingsheng; Tian, Jianghai; Li, Fanchi; Xue, Bin; Hu, Jiahuan; Cheng, Xiaoyu; Li, Jinxin; Shen, Weide; Li, Bing

    2017-01-30

    Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

  1. Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori

    PubMed Central

    Hu, Jingsheng; Tian, Jianghai; Li, Fanchi; Xue, Bin; Hu, Jiahuan; Cheng, Xiaoyu; Li, Jinxin; Shen, Weide; Li, Bing

    2017-01-01

    Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm. PMID:28134300

  2. Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

    PubMed Central

    Ravishankar, Sudha; Ambady, Anisha; Swetha, Rayapadi G.; Anbarasu, Anand; Ramaiah, Sudha; Sambandamurthy, Vasan K.

    2016-01-01

    Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis. PMID:26794499

  3. Induction voidmeter

    DOEpatents

    Anderson, T.T.; Roop, C.J.; Schmidt, K.J.; Brewer, J.

    1983-12-21

    An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; means for applying an AC excitation signal to said input branch; and means for detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.

  4. Induction voidmeter

    DOEpatents

    Anderson, Thomas T.; Roop, Conard J.; Schmidt, Kenneth J.; Brewer, John

    1986-01-01

    An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge circuit, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; applying an AC excitation signal to said input branch; and detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.

  5. Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

    PubMed

    Hong, Y L; Hossler, P A; Calhoun, D H; Meshnick, S R

    1995-08-01

    Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia.

  6. Aminoacyl-tRNA Synthetase Complexes in Evolution

    PubMed Central

    Havrylenko, Svitlana; Mirande, Marc

    2015-01-01

    Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis. PMID:25807264

  7. Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.

    PubMed

    Kumar, Vinay; Yadav, Shailendra; Soumya, Neelagiri; Kumar, Rohit; Babu, Neerupudi Kishore; Singh, Sushma

    2017-03-25

    Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed Km value of 26.3 ± 0.4 mM for l- glutamate and Vmax value of 2.15 ± 0.07 U mg(-1). Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.

  8. Positional isotope exchange analysis of the pantothenate synthetase reaction.

    PubMed

    Williams, LaKenya; Zheng, Renjian; Blanchard, John S; Raushel, Frank M

    2003-05-06

    Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PP(i) must be free to dissociate from the active site upon formation of the pantoyl-adenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.

  9. A Rationally Engineered Misacylating Aminoacyl-Trna Synthetase

    SciTech Connect

    Bullock, T.L.; Rodriguez-Hernandez, A.; Corigliano, E.M.; Perona, J.J.

    2009-05-12

    Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K{sub M} of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA{sup Gln} by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA{sup Gln} by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA{sup Gln} complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

  10. Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.

    PubMed

    Van Dommelen, Anne; Spaepen, Stijn; Vanderleyden, Jozef

    2009-04-01

    Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

  11. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-04-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.

  12. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed Central

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-01-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover. Images PMID:6113590

  13. Mitochondrial aminoacyl-tRNA synthetases in human disease.

    PubMed

    Konovalova, Svetlana; Tyynismaa, Henna

    2013-04-01

    Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential in the process of transferring genetic information from mitochondrial DNA to the complexes of the oxidative phosphorylation system. These synthetases perform an integral step in the initiation of mitochondrial protein synthesis by charging tRNAs with their cognate amino acids. All mtARSs are encoded by nuclear genes, nine of which have recently been described as disease genes for mitochondrial disorders. Unexpectedly, the clinical presentations of these diseases are highly specific to the affected synthetase. Encephalopathy is the most common manifestation but again with gene-specific outcomes. Other clinical presentations include myopathy with anemia, cardiomyopathy, tubulopathy and hearing loss with female ovarian dysgenesis. Here we review the described mutation types and the associated patient phenotypes. The identified mutation spectrum suggests that only mutation types that allow some residual tRNA-charging activity can result in the described mtARS diseases but the molecular mechanisms behind the selective tissue involvement are not currently understood.

  14. Expression of glutamine synthetase in balloon cells: a basis of their antiepileptic role?

    PubMed

    Buccoliero, Anna Maria; Barba, Carmen; Giordano, Flavio; Baroni, Gianna; Genitori, Lorenzo; Guerrini, Renzo; Taddei, Gian Luigi

    2015-01-01

    Glutamine synthetase is an enzyme involved in the clearance of glutamate, the most potent excitatory neurotransmitter. We studied the immunohistochemical expression of glutamine synthetase in neocortical samples from 5 children who underwent surgery for pharmacoresistant epilepsy and a histological diagnosis of focal cortical dysplasia IIb. In all cases, balloon cells, but not dysmorphic neurons, were immunopositive for glutamine synthetase. This finding suggests that balloon cells can be involved in the neutralization of glutamate and play a protective anti-seizure role.

  15. Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase

    SciTech Connect

    Bonnefond, Luc; Frugier, Magali; Touzé, Elodie; Lorber, Bernard; Florentz, Catherine; Giegé, Richard Rudinger-Thirion, Joëlle; Sauter, Claude

    2007-04-01

    Crystals of human mitochondrial tyrosyl-tRNA synthetase lacking the C-terminal S4-like domain diffract to 2.7 Å resolution and are suitable for structure determination. Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA{sup Tyr} charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4{sub 3}2{sub 1}2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 Å resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

  16. Functional expansion of human tRNA synthetases achieved by structural inventions.

    PubMed

    Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-21

    Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions.

  17. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    PubMed

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  18. Assignment of the cysteinyl-tRNA synthetase gene (CARS) to 11p15. 5

    SciTech Connect

    Cruzen, M.E.; Bengtsson, U.; McMahon, J.; Wasmuth, J.J.; Arfin, S.M. )

    1993-03-01

    The attachment of each of the 20 naturally occurring amino acids to their cognate tRNA isoaccepting families is catalyzed by a specific aminoacyl-tRNA synthetase. The structural genes encoding 10 of these enzymes have been assigned to specific human chromosomes. The HARS, LARS, RARS, and TARS genes, encoding histidyl-, leucyl-, arginyl-, and threonyl-tRNA synthetases, respectively, are all located on chromosome 5( 1, 5, 7, 9, 14). The MARS (methionyl-tRNA synthetase), NARS (asparaginyl-tRNA synthetase), VARS (valyl-tRNA synthetase), and WARS (tryptophanyl-tRNA synthetase) genes have been assigned to chromosomes 12, 18, 6, and 14, respectively (3, 4, 6, 8). A gene originally identified as encoding glutaminyl-tRNA synthetase was mapped to chromosome 1q32-q42 (10). However, a recent study suggests that the product of this gene is, in fact, a multifunctional enzyme with both glutamyl- and prolyl-tRNA synthetase activities (2). The fact that 4 of the 10 aminoacyl-tRNA synthetase genes already mapped are located on chromosome 5 may be fortuitous but might also indicate an evolutionary or regulatory relatedness. It is therefore, of interest to map genes encoding other aminoacyl-tRNA synthetases to determine if additional examples of synteny exist. The recent isolation of cDNA and genomic DNA clones for human cysteinyl-tRNA synthetase has now enabled us to map the CARS gene to segment p15.5 on chromosome 11 by fluorescence in situ hybridization.

  19. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.

    PubMed

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Lamers, Wouter H; Chaudhry, Farrukh A; Verlander, Jill W; Weiner, I David

    2016-06-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

  20. Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy.

    PubMed

    Young, D; Fong, D M; Lawlor, P A; Wu, A; Mouravlev, A; McRae, M; Glass, M; Dragunow, M; During, M J

    2014-12-01

    Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94-96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.

  1. Lanthionine Synthetase C-Like 2 Modulates Immune Responses to Influenza Virus Infection

    PubMed Central

    Leber, Andrew; Bassaganya-Riera, Josep; Tubau-Juni, Nuria; Zoccoli-Rodriguez, Victoria; Lu, Pinyi; Godfrey, Victoria; Kale, Shiv; Hontecillas, Raquel

    2017-01-01

    Broad-based, host-targeted therapeutics have the potential to ameliorate viral infections without inducing antiviral resistance. We identified lanthionine synthetase C-like 2 (LANCL2) as a new therapeutic target for immunoinflammatory diseases. To examine the therapeutic efficacy of oral NSC61610 administration on influenza, we infected C57BL/6 mice with influenza A H1N1pdm virus and evaluated influenza-related mortality, lung inflammatory profiles, and pulmonary histopathology. Oral treatment with NSC61610 ameliorates influenza virus infection by down-modulating pulmonary inflammation through the downregulation of TNF-α and MCP-1 and reduction in the infiltration of neutrophils. NSC61610 treatment increases IL10-producing CD8+ T cells and macrophages in the lungs during the resolution phase of disease. The loss of LANCL2 or neutralization of IL-10 in mice infected with influenza virus abrogates the ability of NSC61610 to accelerate recovery and induce IL-10-mediated regulatory responses. These studies validate that oral treatment with NSC61610 ameliorates morbidity and mortality and accelerates recovery during influenza virus infection through a mechanism mediated by activation of LANCL2 and subsequent induction of IL-10 responses by CD8+ T cells and macrophages in the lungs. PMID:28270815

  2. Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

    PubMed Central

    Robertson, J. G.; Sparvero, L. J.; Villafranca, J. J.

    1992-01-01

    Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues. PMID:1303749

  3. Mammalian long-chain acyl-CoA synthetases.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2008-05-01

    Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

  4. Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

    PubMed Central

    Schwartz, Dirk; Grammel, Nicolas; Heinzelmann, Eva; Keller, Ullrich; Wohlleben, Wolfgang

    2005-01-01

    The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release. PMID:16251301

  5. Glutamine versus Ammonia Utilization in the NAD Synthetase Family

    PubMed Central

    Shatalin, Konstantin; Gelfand, Mikhail S.; Osterman, Andrei L.; Sorci, Leonardo

    2012-01-01

    NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural

  6. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

    PubMed Central

    Frago, Susana; Martínez-Júlvez, Marta; Serrano, Ana; Medina, Milagros

    2008-01-01

    Background The prokaryotic FAD synthetase family – a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure. Results Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity. Conclusion For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain. PMID:18811972

  7. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes.

    PubMed

    Frago, Susana; Martínez-Júlvez, Marta; Serrano, Ana; Medina, Milagros

    2008-09-23

    The prokaryotic FAD synthetase family - a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure. Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity. For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain.

  8. Stability of Rat Brain Glutamine Synthetase to Oxygen Toxicity (Oxygen at High Pressure).

    DTIC Science & Technology

    1983-07-01

    Enzyme assays using the gamma-glutamyl transferase method provided estimates of glutamine synthetase activity in rat brain homogenates subjected to a...supports the lack of any connection between convulsions caused by in vivo inhibition of glutamine synthetase and convulsions caused by oxygen toxicity (oxygen at high pressure). (Author)

  9. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

    2008-04-08

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  10. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

    2012-05-22

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  11. Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

    2011-09-06

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  12. Enhancement of lysyl-tRNA synthetase activity in the Enterobacteriaceae

    SciTech Connect

    Hickey, E.W.; Hirshfield, I.

    1987-05-01

    Lysyl-tRNA synthetase (LRS) in E. coli is coded by two genes, one constitutive, and the other inducible; the latter is a cell stress protein. To determine if this system is wide spread in prokaryotes, the inducibility of LRS was first tested in eight members of the Enterobacteriaceae using cultural conditions known to induce the enzyme in E. coli K-12. Uninduced control cultures were grown to an O.D. of 0.2 at 580 nm in a supplemented minimal medium (SMM), pH 7.0 at 37/sup 0/C. Induction stimuli include: growth in SMM with 3mM Gly-L-Leu; growth in SMM as above, but with the initial pH adjusted to 5.0; or growth in Difco AC Broth to early stationary phase with a concomitant drop in the pH of the medium below 5.5. LRS activity was assayed in whole-cell sonic extracts by the aminoacylation of crude E. coli tRNA by /sup 14/C-lysine at pH 7.8 for three minutes. When E. aerogenes, K. pneumoniae, C. freundii, and S. typhimurium were grown in AC Broth, LRS activity was enhanced 2 to 4 fold. The enzyme is induced 2 to 4 fold in C. freundii and S. typhimurium upon growth at pH 5.0, whereas E. coli, K.; pneumoniae, and E. aerogenes show only a 1.5 fold induction. The peptide Gly-L-Leu enhanced LRS activity only in E. coli. LRS was not found to be inducible in S. marcescens, M. morganii, P. mirabilis, or P. vulgaris by any of the stimuli.

  13. An Oxalyl-CoA Synthetase Is Involved in Oxalate Degradation and Aluminum Tolerance.

    PubMed

    Lou, He Qiang; Fan, Wei; Xu, Jia Meng; Gong, Yu Long; Jin, Jian Feng; Chen, Wei Wei; Liu, Ling Yu; Hai, Mei Rong; Yang, Jian Li; Zheng, Shao Jian

    2016-11-01

    Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with Km = 121 ± 8.2 µm and Vmax of 7.7 ± 0.88 µmol min(-1) mg(-1) protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wild-type plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms.

  14. An Oxalyl-CoA Synthetase Is Involved in Oxalate Degradation and Aluminum Tolerance1[OPEN

    PubMed Central

    Fan, Wei; Xu, Jia Meng; Gong, Yu Long; Jin, Jian Feng; Chen, Wei Wei; Liu, Ling Yu; Hai, Mei Rong

    2016-01-01

    Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with Km = 121 ± 8.2 µm and Vmax of 7.7 ± 0.88 µmol min−1 mg−1 protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wild-type plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms. PMID:27650448

  15. Peptide Mapping of Aminoacyl-tRNA Synthetases: Evidence for Internal Sequence Homology in Escherichia coli Leucyl-tRNA Synthetase

    PubMed Central

    Waterson, Robert M.; Konigsberg, William H.

    1974-01-01

    Most aminoacyl-tRNA synthetases contain polypeptide chains of about either 50,000 or 100,000 daltons. Peptide mapping of tryptic, chymotryptic, or Staphylococcus aureus acid protease digests of seryl-tRNA synthetase (100,000, dimer) and leucyl-tRNA synthetase (100,000, monomer) from E. coli was done after selective modification of lysine residues with [14C]succinic anhydride or of methionine residues with [14C]iodoacetate. By use of thin-layer electrophoresis and chromatography on silicagel or cellulose plates followed by radioautography it was possible, depending upon the specific activity of the reagent used, to detect radioactive peptides obtained from as little as l μg of protein. Seryl-tRNA synthetase gave the correct number of tryptic peptides expected for a dimer of identical subunits. Leucyl-tRNA synthetase, on the other hand, gave roughly half the number of radioactive tryptic, chymotryptic, and acid protease peptides expected from the lysine, arginine, and methionine content of the 100,000 monomer. We have interpreted these results as indicating that extensive internal homology exists among lysine- and methionine-containing peptides within the leucyl-tRNA synthetase. The simplest conclusion that can be drawn from these observations is that the NH2- and COOH-terminal halves of leucyl-tRNA synthetase and perhaps other synthetases of 100,000 molecular weight may have evolved through a process of gene duplication and fusion, followed by limited diversification by way of amino-acid substitutions accumulating during evolution. Images PMID:4592690

  16. Circumstantial evidence for a role of glutamine-synthetase in suicide.

    PubMed

    Kalkman, Hans O

    2011-06-01

    Suicide occurs during depression, schizophrenia, diabetes and epilepsy. A common denominator of these disorders is the presence of inflammation. Inflammatory cytokines affect function and expression of the glial enzyme glutamine synthetase and post mortem studies indicate that brain glutamine synthetase function is suppressed in mood disorders and epilepsy. In a study of schizophrenia brains, the expression of glutamine synthetase was reduced in those cases where the cause of death was suicide. The glycogen synthase kinase 3 (GSK3) inhibitor, lithium, which has a proven efficacy against suicide, increased in an animal experiment the expression of glutamine synthetase. Based on these data one could reason that suicide may be prevented by centrally acting GSK3 inhibitors. However, since inhibition of glutamine synthetase may lead to a deficit in glutamine and as consequence a GABA and glutamate deficit, even simple food supplementation with glutamine might help to reduce suicide.

  17. Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity

    PubMed Central

    Gibson, K.; Harris, P.

    1972-01-01

    The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of 14C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[14C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected. PMID:5071178

  18. Inhibition of rabbit gastric glucosamine synthetase activity by Cu2+, Zn2+ and Se4+.

    PubMed

    Fujita, T; Sakuma, S; Takahashi, K; Bohtani, Y; Nishida, H; Fujimoto, Y

    1997-05-01

    The effects of Fe2+, Cu2+, Zn2+ and Se4+ on the activity of glucosamine synthetase, the rate-limiting enzyme of mucus synthesis, in rabbit gastric corporal mucosa were examined. Cu2+, Zn2+ and Se4+ inhibited the glucosamine synthetase activity at concentrations ranging from 1 to 10 microM (Cu2+, 8-98% inhibition; Zn2+, 10-99% inhibition; Se4+, 32-89% inhibition). The inhibitory effects of these three ions were much stronger than that of UDP-N-acetylglúcosamine known as a representative inhibitor of the glucosamine synthetase activity (10 microM, 52% inhibition). Fe2+ had no significant effect on the glucosamine synthetase activity up to 100 microM. These results suggest that Cu2+, Zn2+ and Se4+ can be potent inhibitors of gastric glucosamine synthetase activity.

  19. The evolution of Class II Aminoacyl-tRNA synthetases and the first code.

    PubMed

    Smith, Temple F; Hartman, Hyman

    2015-11-30

    Class II Aminoacyl-tRNA synthetases are a set of very ancient multi domain proteins. The evolution of the catalytic domain of Class II synthetases can be reconstructed from three peptidyl-hairpins. Further evolution from this primordial catalytic core leads to a split of the Class II synthetases into two divisions potentially associated with the operational code. The earliest form of this code likely coded predominantly Glycine (Gly), Proline (Pro), Alanine (Ala) and "Lysine"/Aspartic acid (Lys/Asp). There is a paradox in these synthetases beginning with a hairpin structure before the Genetic Code existed. A resolution is found in the suggestion that the primordial Aminoacyl synthetases formed in a transition from a Thioester world to a Phosphate ester world. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    PubMed Central

    Pyne, C; Bognar, A L

    1992-01-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products. Images PMID:1548226

  1. Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver.

    PubMed

    Cabrero, C; Puerta, J; Alemany, S

    1987-12-30

    Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.

  2. Induction synchrotron

    NASA Astrophysics Data System (ADS)

    Takayama, Ken; Kishiro, Junichi

    2000-08-01

    A novel proton synchrotron employing induction cells instead of radio frequency cavities is proposed. The major feature of the barrier bucket acceleration, where acceleration and longitudinal focusing are independently achieved is theoretically discussed with the help of multi-particle simulations. It is proved that barrier bucket acceleration allows ultimate use of longitudinal phase-space and is quite effective to substantially increase the beam intensity in synchrotrons. Engineering aspects of key devices to realize the novel synchrotron, a ferri/ferro-magnetic material loaded induction cell and a modulator being rapidly switched in synchronization with beam acceleration are described in detail. The idea is applied to an existing machine (the KEK 12 GeV-PS) and high-intensity proton rings such as JHF, ESS, and SNS and their predicted improvement in machine performance is given with numerical values for each case.

  3. Induction machine

    SciTech Connect

    Owen, W.H.

    1980-10-14

    A polyphase rotary induction machine for use as a motor or generator utilizes, a single rotor assembly having two series connected sets of rotor windings, a first stator winding disposed around the first rotor winding and means for controlling the current induced in one set of the rotor windings compared to the current induced in the other set of the rotor windings. The rotor windings may be wound rotor windings or squirrel cage windings.

  4. Induction machine

    DOEpatents

    Owen, Whitney H.

    1980-01-01

    A polyphase rotary induction machine for use as a motor or generator utilizing a single rotor assembly having two series connected sets of rotor windings, a first stator winding disposed around the first rotor winding and means for controlling the current induced in one set of the rotor windings compared to the current induced in the other set of the rotor windings. The rotor windings may be wound rotor windings or squirrel cage windings.

  5. Novel insights into regulation of asparagine synthetase in conifers.

    PubMed

    Canales, Javier; Rueda-López, Marina; Craven-Bartle, Blanca; Avila, Concepción; Cánovas, Francisco M

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

  6. Novel Insights into Regulation of Asparagine Synthetase in Conifers

    PubMed Central

    Canales, Javier; Rueda-López, Marina; Craven-Bartle, Blanca; Avila, Concepción; Cánovas, Francisco M.

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed. PMID:22654888

  7. Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase

    PubMed Central

    Alderwick, Luke J; Lloyd, Georgina S; Lloyd, Adrian J; Lovering, Andrew L; Eggeling, Lothar; Besra, Gurdyal S

    2011-01-01

    Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate (pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase. PMID:21045009

  8. Mammalian folylpoly-. gamma. -glutamate synthetase. 3. Specificity for folate analogues

    SciTech Connect

    George, S.; Cichowicz, D.J.; Shane, B.

    1987-01-27

    A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folypolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the ..gamma..-carboxyl of the terminal glutamate in the correct position for catalysis. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates. Other folate derivatives with tetrahedral chemistry replacing the peptide bond, such as pteroyl-..gamma..-glutamyl-(psi,CH/sub 2/-NH)-glutamate, retain affinity for the protein but are considerably less effective inhibitors than the ornithine derivatives. Enzyme activity was assayed using (/sup 14/C)glutamate.

  9. MANAGEMENT OF A PATIENT WITH HOLOCARBOXYLASE SYNTHETASE DEFICIENCY

    PubMed Central

    Van Hove, Johan LK; Josefsberg, Sagi; Freehauf, Cynthia; Thomas, Janet A.; Thuy, Le Phuc; Barshop, Bruce A.; Woontner, Michael; Mock, Donald M; Chiang, Pei-Wen; Spector, Elaine; Meneses-Morales, Iván; Cervantes-Roldán, Rafael; León-Del-Río, Alfonso

    2009-01-01

    We investigated in a patient with holocarboxylase synthetase deficiency, the relation between the biochemical and genetic factors of the mutant protein with the pharmacokinetic factors of successful biotin treatment. A girl exhibited abnormal skin at birth, and developed in the first days of life neonatal respiratory distress syndrome and metabolic abnormalities diagnostic of multiple carboxylase deficiency. Enzyme assays showed low carboxylase activities. Fibroblast analysis showed poor incorporation of biotin into the carboxylases, and low transfer of biotin by the holocarboxylase synthetase enzyme. Kinetic studies identified an increased Km but a preserved Vmax. Mutation analysis showed the child to be a compound heterozygote for a new nonsense mutation Q379X and for a novel missense mutation Y663H. This mutation affects a conserved amino acid, which is located the most 3′ of all recorded missense mutations thus far described, and extends the region of functional biotin interaction. Treatment with biotin 100 mg/day gradually improved the biochemical abnormalities in blood and in cerebrospinal fluid, corrected the carboxylase enzyme activities, and provided clinical stability and a normal neurodevelopmental outcome. Plasma concentrations of biotin were increased to more than 500 nM, thus exceeding the increased Km of the mutant enzyme. At these pharmacological concentrations, the CSF biotin concentration was half the concentration in blood. Measuring these pharmacokinetic variables can aid in optimizing treatment, as individual tailoring of dosing to the needs of the mutation may be required. PMID:18974016

  10. Management of a patient with holocarboxylase synthetase deficiency.

    PubMed

    Van Hove, Johan L K; Josefsberg, Sagi; Freehauf, Cynthia; Thomas, Janet A; Thuy, Le Phuc; Barshop, Bruce A; Woontner, Michael; Mock, Donald M; Chiang, Pei-Wen; Spector, Elaine; Meneses-Morales, Iván; Cervantes-Roldán, Rafael; León-Del-Río, Alfonso

    2008-12-01

    We investigated in a patient with holocarboxylase synthetase deficiency, the relation between the biochemical and genetic factors of the mutant protein with the pharmacokinetic factors of successful biotin treatment. A girl exhibited abnormal skin at birth, and developed in the first days of life neonatal respiratory distress syndrome and metabolic abnormalities diagnostic of multiple carboxylase deficiency. Enzyme assays showed low carboxylase activities. Fibroblast analysis showed poor incorporation of biotin into the carboxylases, and low transfer of biotin by the holocarboxylase synthetase enzyme. Kinetic studies identified an increased Km but a preserved Vmax. Mutation analysis showed the child to be a compound heterozygote for a new nonsense mutation Q379X and for a novel missense mutation Y663H. This mutation affects a conserved amino acid, which is located the most 3' of all recorded missense mutations thus far described, and extends the region of functional biotin interaction. Treatment with biotin 100mg/day gradually improved the biochemical abnormalities in blood and in cerebrospinal fluid (CSF), corrected the carboxylase enzyme activities, and provided clinical stability and a normal neurodevelopmental outcome. Plasma concentrations of biotin were increased to more than 500 nM, thus exceeding the increased Km of the mutant enzyme. At these pharmacological concentrations, the CSF biotin concentration was half the concentration in blood. Measuring these pharmacokinetic variables can aid in optimizing treatment, as individual tailoring of dosing to the needs of the mutation may be required.

  11. Purification and properties of the dihydrofolate synthetase from Serratia indica.

    PubMed

    Ikeda, M; Kazuo, I

    1976-01-01

    The dihydrofolate synthetase (EC 6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg2+ and K+ as cofactors. gamma-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of AD, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP by the following equation: 7,8-Dihydropteroic acid ml-Glutamic acid matp Mg2+, K+ leads to Dihydrofolic acid + ADP + Pi. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP).

  12. Crystallization and preliminary X-ray analysis of RsbS from Moorella thermoacetica at 2.5 A resolution.

    PubMed

    Quin, Maureen; Newman, Joseph; Firbank, Susan; Lewis, Richard J; Marles-Wright, Jon

    2008-03-01

    The thermophilic bacterium Moorella thermoacetica possesses an rsb operon that is related to the genetic locus common to many Gram-positive bacteria that regulates the activity of the stress-responsive sigma factor sigma(B). One of the gene products of this operon is RsbS, a single STAS-domain protein that is a component of higher order assemblies in Bacillus subtilis known as 'stressosomes'. It is expected that similar complexes are found in M. thermoacetica, but in this instance regulating the biosynthesis of cyclic di-GMP, a ubiquitous secondary messenger. Selenomethionine-labelled MtRsbS protein was crystallized at room temperature using the hanging-drop vapour-diffusion method. Crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.07, b = 60.52, c = 89.28 A, diffracted to 2.5 A resolution on beamline I04 of the Diamond Light Source. The selenium substructure was solved using SHELX and it is believed that this represents the first reported ab initio crystal structure to be solved using diffraction data collected at DLS.

  13. Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

    PubMed

    Savilov, P N; Yakovlev, V N

    2016-01-01

    Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

  14. Encapsulation of glutamine synthetase in mouse erythrocytes: a new procedure for ammonia detoxification.

    PubMed

    Kosenko, Elena A; Venediktova, Natalia I; Kudryavtsev, Andrey A; Ataullakhanov, Fazoil I; Kaminsky, Yury G; Felipo, Vicente; Montoliu, Carmina

    2008-12-01

    There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 +/- 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations.

  15. Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing

    SciTech Connect

    Freytag, S.O.; Beaudet, A.L.; Bock, H.G.O.; O'Brien, W.E.

    1984-10-01

    The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

  16. Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

    PubMed

    Jeitner, Thomas M; Battaile, Kevin; Cooper, Arthur J L

    2015-12-01

    The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

  17. Isolation of a cDNA clone for human threonyl-tRNA synthetase

    SciTech Connect

    Kontis, K.J.; Arfin, S.M.

    1989-05-01

    A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA /lambda/gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

  18. Organizing pneumonia as the first manifestation of anti-synthetase syndrome.

    PubMed

    Priyangika, S M Thanuja Nilushi; Karunarathna, W G S G; Liyanage, Isurujith; Gunawardana, Methsala; Udumalgala, Sumeda; Rosa, Chamith; Kulatunga, Aruna

    2016-06-02

    Anti-synthetase syndrome associated interstitial lung disease can occur either simultaneously, before, or after the development of polymyositis/dermatomyositis. Histology of interstitial lung disease can be nonspecific interstitial pneumonia, usual interstitial pneumonia, diffuse alveolar damage, organizing pneumonia. Organizing pneumonia associated anti-synthetase syndrome is a rare finding especially as the first manifestation. We report a 41 year old male patient who presented with organizing pneumonia and 2 years following the onset, developed polymyositis with anti-JO-1 antibody positivity. It is important to screen patients with organizing pneumonia for anti-synthetase syndrome which can be manifested later.

  19. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  20. Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency.

    PubMed Central

    Burri, B J; Sweetman, L; Nyhan, W L

    1985-01-01

    Holocarboxylase synthetase activity has been determined in fibroblasts of seven patients with the neonatal form of biotin-responsive multiple carboxylase deficiency. The normal Km for biotin was 15 +/- 3 nmol/l, while in the patients the values ranged from 48 to 1,062 nmol/l. The mean maximum velocity was 27% of normal. Differences among the values obtained for the Km for biotin and the heat stability of holocarboxylase synthetase suggested that the patients studied represented at least four distinct variants at the holocarboxylase synthetase locus. PMID:3920902

  1. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

    PubMed

    Mashek, Douglas G; Bornfeldt, Karin E; Coleman, Rosalind A; Berger, Johannes; Bernlohr, David A; Black, Paul; DiRusso, Concetta C; Farber, Steven A; Guo, Wen; Hashimoto, Naohiro; Khodiyar, Varsha; Kuypers, Frans A; Maltais, Lois J; Nebert, Daniel W; Renieri, Alessandra; Schaffer, Jean E; Stahl, Andreas; Watkins, Paul A; Vasiliou, Vasilis; Yamamoto, Tokuo T

    2004-10-01

    By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

  2. Adapalene/Benzoyl Peroxide Gel 0.3%/2.5%: A Safe and Effective Acne Therapy in All Skin Phototypes.

    PubMed

    Alexis, Andrew F; Cook-Bolden, Fran E; York, J P

    2017-06-01

    BACKGROUND: Acne affects individuals of all races and ethnicities; however, lighter and darker skin phototypes face different treatment challenges that may affect treatment response and tolerability. This analysis investigated possible differences in the efficacy and safety of the fixed dose combination of 0.3% adapalene with 2.5% benzoyl peroxide (A/BPO gel 0.3%/2.5%) in subjects with Fitzpatrick Skin Types (FST) I-VI.

    METHODS: This was a post-hoc analysis of a Phase 3, multicenter, randomized, double-blind, parallel-group study of moderate to severe acne in subjects with FST I-VI. Subjects received A/BPO gel 0.3%/2.5%, A/BPO gel 0.1%/2.5% (benchmark), or vehicle, once daily for 12 weeks. Efficacy measurements included success rate (IGA of Clear or Almost Clear), change in inflammatory and noninflammatory lesions from baseline to week 12, safety, and tolerability. The intent to treat (ITT) and safety populations were analyzed. Demographics and disposition were analyzed with descriptive statistics; categorical variables by frequency and percentage; and continuous variables with means, medians, minimum, maximum, and standard deviations.

    RESULTS: The A/BPO gel 0.3%/2.5% treatment group included 128 subjects with FST I-III, and 89 subjects with FST IV-VI. At week 12, A/BPO gel 0.3%/2.5% was safe, tolerable, and significantly superior to vehicle for all FST and severity groups in inflammatory and noninflammatory lesion reduction (P less than equal to .05). Compared to baseline, 32% of subjects with FST I-III were clear or almost clear, compared to 7% in the vehicle group (P=.001). In FST IV-VI, 28% of subjects were clear or almost clear, compared to 15% for vehicle (P=NS). In all treatment groups and skin phototypes, week 12 tolerability scores were similar to baseline scores, and tolerability scores for most subjects of all skin phototypes were "none" or "mild" for all measures.

    SUMMARY: We report that the fixed dose combination of A/BPO gel 0

  3. Aminoacyl-tRNA synthetases in medicine and disease

    PubMed Central

    Yao, Peng; Fox, Paul L

    2013-01-01

    Aminoacyl-tRNA synthetases (ARSs) are essential and ubiquitous ‘house-keeping’ enzymes responsible for charging amino acids to their cognate tRNAs and providing the substrates for global protein synthesis. Recent studies have revealed a role of multiple ARSs in pathology, and their potential use as pharmacological targets and therapeutic reagents. The ongoing discovery of genetic mutations in human ARSs is increasing exponentially and can be considered an important determinant of disease etiology. Several chemical compounds target bacterial, fungal and human ARSs as antibiotics or disease-targeting medicines. Remarkably, ongoing exploration of noncanonical functions of ARSs has shown important contributions to control of angiogenesis, inflammation, tumourigenesis and other important physiopathological processes. Here, we summarize the roles of ARSs in human diseases and medicine, focusing on the most recent and exciting discoveries. PMID:23427196

  4. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

    PubMed Central

    Mirando, Adam C.; Francklyn, Christopher S.; Lounsbury, Karen M.

    2014-01-01

    In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis. PMID:25535072

  5. Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis

    PubMed Central

    Donini, Stefano; Garavaglia, Silvia; Ferraris, Davide M.; Miggiano, Riccardo; Mori, Shigetarou; Shibayama, Keigo

    2017-01-01

    Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest. PMID:28419153

  6. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

    PubMed Central

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2017-01-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells. PMID:25645621

  7. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

    PubMed

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2015-05-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

  8. Reduced glutamine synthetase activity plays a role in control of photosynthetic responses to high light in barley leaves.

    PubMed

    Brestic, Marian; Zivcak, Marek; Olsovska, Katarina; Shao, Hong-Bo; Kalaji, Hazem M; Allakhverdiev, Suleyman I

    2014-08-01

    The chloroplastic glutamine synthetase (GS, EC 6.3.1.2) activity was previously shown to be the limiting step of photorespiratory pathway. In our experiment, we examined the photosynthetic high-light responses of the GS2-mutant of barley (Hordeum vulgare L.) with reduced GS activity, in comparison to wild type (WT). The biophysical methods based on slow and fast chlorophyll fluorescence induction, P700 absorbance, and gas exchange measurements were employed. Despite the GS2 plants had high basal fluorescence (F0) and low maximum quantum yield (Fv/Fm), the CO2 assimilation rate, the PSII and PSI actual quantum yields were normal. On the other hand, in high light conditions the GS2 had much higher non-photochemical quenching (NPQ), caused both by enhanced capacity of energy-dependent quenching and disconnection of PSII antennae from reaction centers (RC). GS2 leaves also maintained the PSII redox poise (QA(-)/QA total) at very low level; probably this was reason why the observed photoinhibitory damage was not significantly above WT. The analysis of fast chlorophyll fluorescence induction uncovered in GS2 leaves substantially lower RC to antenna ratio (RC/ABS), low PSII/PSI ratio (confirmed by P700 records) as well as low PSII excitonic connectivity.

  9. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    PubMed

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

    2015-07-01

    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  10. Redesigning the stereospecificity of tyrosyl-tRNA synthetase.

    PubMed

    Simonson, Thomas; Ye-Lehmann, Shixin; Palmai, Zoltan; Amara, Najette; Wydau-Dematteis, Sandra; Bigan, Erwan; Druart, Karen; Moch, Clara; Plateau, Pierre

    2016-02-01

    D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

  11. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    SciTech Connect

    Rubin, Edward M.

    1980-01-01

    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy/sup +/ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy/sup +/ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy/sup +/ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  12. Activity of formylphosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

    Jahansouz, H.; Kofron, J.L.; Smithers, G.W.; Himes, R.H.; Reed, G.H.

    1986-05-01

    Formylphosphate (FP), a putative enzyme-bound intermediate in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase, was synthesized from formylfluoride and Pi. Measurement of hydrolysis rates by /sup 31/P NMR showed that FP is very unstable with a half-life of 48 min at 20/sup 0/C and pH 7. At pH 7 hydrolysis occurs with O-P bond cleavage as shown by /sup 18/O incorporation from /sup 18/O-H/sub 2/O into Pi. The substrate activity of FP was tested in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase isolated from Clostridium cylindrosporum. MgATP + H/sub 4/folate + HCOO/sup -/ in equilibrium MgADP + Pi +N/sup 10/-formylH/sub 4/folate FP supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formylH/sub 4/folate is produced from H/sub 4/-folate and FP but only if ADP is present, and ATP is produced from FP and ADP but only if H/sub 4/folate is present. The requirements for H/sub 4/folate in the synthesis of ATP from ADP and FP and for ADP in the synthesis of N/sup 10/-formylH/sub 4/folate from FP and H/sub 4/folate, are consistent with past kinetic and isotope exchange studies which showed that the reaction proceeds by a sequential mechanism and that all three substrates must be present for any reaction to occur.

  13. Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei.

    PubMed

    Katoh, Hiroki; Tamaki, Hideyuki; Tokutake, Yuka; Hanada, Satoshi; Chohnan, Shigeru

    2013-04-01

    Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

  14. Identification and functional characterization of a novel bacterial type asparagine synthetase A: a tRNA synthetase paralog from Leishmania donovani.

    PubMed

    Manhas, Reetika; Tripathi, Pankaj; Khan, Sameena; Sethu Lakshmi, Bhavana; Lal, Shambhu Krishan; Gowri, Venkatraman Subramanian; Sharma, Amit; Madhubala, Rentala

    2014-04-25

    Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.

  15. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase.

    PubMed

    Bowles, Tawnya L; Kim, Randie; Galante, Joseph; Parsons, Colin M; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J

    2008-10-15

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by approximately 50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

  16. Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus.

    PubMed

    Miao, G H; Hirel, B; Marsolier, M C; Ridge, R W; Verma, D P

    1991-01-01

    A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

  17. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

    PubMed Central

    Bowles, Tawnya L.; Kim, Randie; Galante, Joseph; Parsons, Colin M.; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J.

    2009-01-01

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is under-expressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by ∼50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed. PMID:18661517

  18. Purification and determination of glutamine synthetase by high-performance immunoaffinity chromatography.

    PubMed

    Alhama, J; López-Barea, J; Toribio, F; Roldán, J M

    1992-01-10

    High-performance immunoaffinity chromatography (HPIAC) with anti-glutamine synthetase polyclonal antibodies bound to epoxy-activated silica was used to purify and determine this enzyme from the cyanobacterium Synechocystis. A single-step HPIAC procedure with cell-free extracts yielded electroporetically homogeneous glutamine synthetase. In the determination of glutamine synthetase by HPIAC a linear response in the range 10-60 micrograms of enzyme was observed. Recoveries of 70% of the loaded enzymatic activity and 100% of protein were obtained. The determination of glutamine synthetase protein by HPIAC was compared with that obtained by rocket immunoelectrophoresis. The chromatographic method is proposed as a possible alternative to other immunochemical quantitative techniques, particularly when non-limiting amounts of samples are available.

  19. Preparation and cross-reactivity of anti-avian glutamine synthetase antibody.

    PubMed

    Smith, D D; Vorhaben, J E; Campbell, J W

    1983-04-01

    Rabbit antibody to chicken liver mitochondrial glutamine synthetase was purified by immunoaffinity chromatography for analysis of the immunological relatedness of vertebrate glutamine synthetases. The antibody cross-reacted with enzymes from representatives of all five vertebrate classes, indicating a high degree of evolutionary conservatism in the structure of the enzymes. A unique aspect of the immunological similarity of these enzymes is that it exists between cytosolic and mitochondrial enzymes which are, in general, immunologically distinct. The antibody did not cross-react with two insect glutamine synthetases. Compositional difference indices, calculated from the amino acid compositions of glutamine synthetases from several species, gave a mean estimate of over 80% sequence homology for the vertebrate enzymes. The avian mitochondrial enzyme gave a mean 78% homology with the mammalian cytosolic enzyme.

  20. Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

    1974-01-01

    Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.

  1. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  2. Severe holocarboxylase synthetase deficiency with incomplete biotin responsiveness resulting in antenatal insult in samoan neonates.

    PubMed

    Wilson, Callum J; Myer, Michael; Darlow, Brian A; Stanley, Thorsten; Thomson, Glen; Baumgartner, E Regula; Kirby, Denise M; Thorburn, David R

    2005-07-01

    We describe 7 Polynesian babies with a unique severe form of holocarboxylase synthetase deficiency characterized by antenatal growth retardation, subependymal cysts, only partial response to biotin, and a poor outcome.

  3. Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases.

    PubMed Central

    Jakubowski, H; Fersht, A R

    1981-01-01

    Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetase operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pre-transfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed. PMID:7024910

  4. Functional expansion of human tRNA synthetases achieved by structural inventions

    PubMed Central

    Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions. PMID:19932696

  5. Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

    1974-01-01

    Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.

  6. Diffuse glutamine synthetase overexpression restricted to areas of peliosis in a β-catenin-activated hepatocellular adenoma: a potential pitfall in glutamine synthetase interpretation.

    PubMed

    Berry, Ryan S; Gullapalli, Rama R; Wu, Jin; Morris, Katherine; Hanson, Joshua A

    2014-08-01

    Hepatocellular adenomas have recently been classified into four subtypes based on molecular findings: hepatocyte nuclear factor 1α (HNF1α) inactivated, inflammatory/telangiectatic, β-catenin activated, and unclassifiable. β-catenin-activated adenomas have the potential for malignant transformation and are thus important to recognize. Diffuse glutamine synthetase immunohistochemical positivity has been shown to be a reliable surrogate marker for β-catenin activation, though variations in staining patterns may be difficult to interpret. We report a case of a peliotic adenoma that was morphologically consistent with a β-catenin wild-type hepatocellular adenoma but harbored a β-catenin mutation by molecular analysis. The tumor lacked nuclear β-catenin positivity and demonstrated a hitherto undescribed pattern of glutamine synthetase overexpression restricted to areas of peliosis with mostly negative staining in non-peliotic areas. This pattern was initially interpreted as physiologic and may represent a potential pitfall in glutamine synthetase interpretation.

  7. Lack of protective effect of thromboxane synthetase inhibitor (CGS-13080) on single dose radiated canine intestine.

    PubMed

    Barter, J F; Marlow, D; Kamath, R K; Harbert, J; Torrisi, J R; Barnes, W A; Potkul, R K; Newsome, J T; Delgado, G

    1991-03-01

    The effect of a thromboxane A2 synthetase inhibitor (CGS-13080) on canine intestine was studied using a single dose of radiation, and radioactive microspheres were used to determine resultant blood flow. Thromboxane A2 causes vasospasm and platelet aggregation and may play a dominant role in radiation injury. However, there was no effect on the intestinal blood flow diminution occurring after radiation in this laboratory model using this thromboxane A2 synthetase inhibitor.

  8. Lack of protective effect of thromboxane synthetase inhibitor (CGS-13080) on single dose radiated canine intestine

    SciTech Connect

    Barter, J.F.; Marlow, D.; Kamath, R.K.; Harbert, J.; Torrisi, J.R.; Barnes, W.A.; Potkul, R.K.; Newsome, J.T.; Delgado, G. )

    1991-03-01

    The effect of a thromboxane A2 synthetase inhibitor (CGS-13080) on canine intestine was studied using a single dose of radiation, and radioactive microspheres were used to determine resultant blood flow. Thromboxane A2 causes vasospasm and platelet aggregation and may play a dominant role in radiation injury. However, there was no effect on the intestinal blood flow diminution occurring after radiation in this laboratory model using this thromboxane A2 synthetase inhibitor.

  9. The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence.

    PubMed

    Avila-Ospina, Liliana; Marmagne, Anne; Talbotec, Joël; Krupinska, Karin; Masclaux-Daubresse, Céline

    2015-04-01

    Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions.

  10. Recurrent seizures and brain pathology after inhibition of glutamine synthetase in the hippocampus in rats.

    PubMed

    Eid, Tore; Ghosh, Arko; Wang, Yue; Beckström, Henning; Zaveri, Hitten P; Lee, Tih-Shih W; Lai, James C K; Malthankar-Phatak, Gauri H; de Lanerolle, Nihal C

    2008-08-01

    An excess of extracellular glutamate in the hippocampus has been linked to the generation of recurrent seizures and brain pathology in patients with medically intractable mesial temporal lobe epilepsy (MTLE). However, the mechanism which results in glutamate excess in MTLE remains unknown. We recently reported that the glutamate-metabolizing enzyme glutamine synthetase is deficient in the hippocampus in patients with MTLE, and we postulated that this deficiency is critically involved in the pathophysiology of the disease. To further explore the role of glutamine synthetase in MTLE we created a novel animal model of hippocampal glutamine synthetase deficiency by continuous (approximately 28 days) microinfusion of methionine sulfoximine (MSO: 0.625 to 2.5 microg/h) unilaterally into the hippocampus in rats. This treatment led to a deficiency in hippocampal glutamine synthetase activity by 82-97% versus saline. The majority (>95%) of the MSO-treated animals exhibited recurrent seizures that continued for several weeks. Some of the MSO-treated animals exhibited neuropathological features that were similar to mesial temporal sclerosis, such as hippocampal atrophy and patterned loss of hippocampal neurons. However, many MSO-treated animals displayed only minimal injury to the hippocampus, with no clear evidence of mesial temporal sclerosis. These findings support the hypothesis that a deficiency in hippocampal glutamine synthetase causes recurrent seizures, even in the absence of classical mesial temporal sclerosis, and that restoration of glutamine synthetase may represent a novel approach to therapeutic intervention in this disease.

  11. CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

    PubMed Central

    Chang, Yu-Fang; Carman, George M.

    2008-01-01

    CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C. PMID:18439916

  12. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    SciTech Connect

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  13. Pivotal role of glutamine synthetase in ammonia detoxification.

    PubMed

    Hakvoort, Theodorus B M; He, Youji; Kulik, Wim; Vermeulen, Jacqueline L M; Duijst, Suzanne; Ruijter, Jan M; Runge, Jurgen H; Deutz, Nicolaas E P; Koehler, S Eleonore; Lamers, Wouter H

    2017-01-01

    Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that ∼35% of this ammonia is detoxified by hepatic GS and ∼35% by urea-cycle enzymes, while ∼30% is not cleared by the liver, independent of portal ammonia concentrations ≤2 mmol/L. Using both genetic (GS-knockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is ∼160 μmol/hour in control mice. Using stable isotopes, we show that disposal of glutamine-bound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from ∼7% in methionine sulfoximine-treated mice to ∼500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on

  14. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do

  15. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

  16. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.

    PubMed

    Verlander, Jill W; Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E; Weiner, I David

    2013-09-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.

  17. Homology modeling and molecular docking studies of Bacillomycin and Iturin synthetases with novel ligands for the production of therapeutic lipopeptides.

    PubMed

    Eswari, Jujjavarapu Satya; Dhagat, Swasti; Kaser, Shubham; Tiwari, Anoop

    2017-08-15

    Lipopeptide synthetases play an important role in the production of lipopeptides. Lipopeptides are molecules made up of peptides and fatty acid moieties and have shown to have a broad range of antimicrobial activity. As infectious diseases have caused severe health problems mainly resulting from the development of antibiotic resistant strains of disease causing microorganisms there is a need of alternatives to antibiotics. The lipopeptide synthetase of the corresponding lipopeptides can be used as templates to design these as drugs using computational techniques. The objective of this study was homology modeling and molecular docking of two lipopeptide synthetases, bacillomycin D synthetase and iturin A synthetase, with their ligands as a means of drug design. Schrödinger software was used for homology modeling and molecular docking. After the identification of ligands, molecular docking of these ligands with the lipopeptide (bacillomycin and iturin) synthetases was performed. The docking was tested on the parameters of docking score and glide energy. 5 out of 21 ligands were found to dock with bacillomycin D synthetase whereas 8 out of 20 ligands docked with the iturin A synthetase. The knowledge of the docking sites and docking characteristics of the lipopeptide synthetases mentioned in the paper with the ligands can provide advantages of high speed and reliability, reduced costs on chemicals and experiments and the ethical issues concerned with the use of animal models for screening of drug toxicity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia

    PubMed Central

    Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E.; Weiner, I. David

    2013-01-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+-free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism. PMID:23804452

  19. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    PubMed

    Theron, A; Roth, R L; Hoppe, H; Parkinson, C; van der Westhuyzen, C W; Stoychev, S; Wiid, I; Pietersen, R D; Baker, B; Kenyon, C P

    2017-01-01

    Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  20. The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2

    SciTech Connect

    Webb, G.C.; Vaska, V.L.; Ford, J.H.

    1995-12-10

    Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxoprolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization. 16 refs., 2 figs.

  1. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

    PubMed Central

    Bender, R A; Janssen, K A; Resnick, A D; Blumenberg, M; Foor, F; Magasanik, B

    1977-01-01

    The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting. PMID:14104

  2. Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis

    PubMed Central

    Binz, Tina M.; Maffioli, Sonia I.; Sosio, Margherita; Donadio, Stefano; Müller, Rolf

    2010-01-01

    The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering. PMID:20710026

  3. Novel acyl-CoA synthetase in adrenoleukodystrophy target tissues.

    PubMed

    Moriya-Sato, A; Hida, A; Inagawa-Ogashiwa, M; Wada, M R; Sugiyama, K; Shimizu, J; Yabuki, T; Seyama, Y; Hashimoto, N

    2000-12-09

    X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by demyelination of white matter. The X-ALD gene product adrenoleukodystrophy protein (ALDP) is expressed broadly among various tissues. However, deficiency of functional ALDP exclusively impairs brain, adrenal gland, and testis. Thus, loss of ALDP function is assumed to involve inactivation of a putative mediating factor that functions in a tissue-specific manner. Here we cloned a mouse cDNA encoding a novel protein, Lipidosin, that possesses long-chain acyl-CoA synthetase (LCAS) activity. Lipidosin is expressed exclusively in mouse brain, adrenal gland, and testis, which are affected by X-ALD. LCAS activity of Lipidosin was diminished by mutation of conserved amino acids within the AMP-binding domain. Mutation of the Drosophila homologue of Lipidosin has been reported to cause neuronal degeneration. Thus, Lipidosin may mediate the link between ALDP dysfunction and the impairment of fatty acid metabolism in X-ALD. Copyright 2000 Academic Press.

  4. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.

  5. Structural Biology of Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Gulick, Andrew M.

    2016-01-01

    Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

  6. The prokaryotic FAD synthetase family: a potential drug target.

    PubMed

    Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

    2013-01-01

    Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

  7. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    SciTech Connect

    Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

    1986-11-01

    The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of (6-/sup 3/H)deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of (6-/sup 3/H)deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells.

  8. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  9. The aminoacyl-tRNA synthetases of Drosophila melanogaster

    PubMed Central

    Lu, Jiongming; Marygold, Steven J; Gharib, Walid H; Suter, Beat

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field. PMID:26761199

  10. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    SciTech Connect

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  11. Versatility of acyl-acyl carrier protein synthetases

    DOE PAGES

    Beld, Joris; Finzel, Kara; Burkart, Michael D.

    2014-10-09

    The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

  12. The complex evolutionary history of aminoacyl-tRNA synthetases

    PubMed Central

    Chaliotis, Anargyros; Vlastaridis, Panayotis; Mossialos, Dimitris; Ibba, Michael; Becker, Hubert D.

    2017-01-01

    Abstract Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code. PMID:28180287

  13. Small Alarmone Synthetases as novel bacterial RNA-binding proteins.

    PubMed

    Hauryliuk, Vasili; Atkinson, Gemma C

    2017-08-18

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as a RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA:RelQ complex. RelQ's allosteric regulator, pppGpp, destabilizes RNA binding and activates RelQ's enzymatic activity. Since SAS enzymes are widely distributed in bacteria, and, as it has been discovered recently, are also mobilized by phages (Dedrick et al. 2017), RNA binding to SAS is could be a wide-spread mechanism. The initial discovery raises numerous questions regarding RNA-binding function of the SAS enzymes: What is the molecular mechanism underlying the incompatibility of RNA:SAS complex formation with pppGpp binding and (p)ppGpp synthesis? What are the RNA targets in living cells? What is the regulatory output of the system - (p)ppGpp synthesis, modulation of RNA structure and function, or both?

  14. Holocarboxylase synthetase: correlation of protein localisation with biological function.

    PubMed

    Bailey, L M; Wallace, J C; Polyak, S W

    2010-04-01

    Holocarboxylase synthetase (HCS) governs the cellular fate of the essential micronutrient biotin (Vitamin H or B7). HCS is responsible for attaching biotin onto the biotin-dependent enzymes that reside in the cytoplasm and mitochondria. Evidence for an alternative role, viz the regulation of gene expression, has also been reported. Recent immunohistochemical studies reported HCS is primarily nuclear, inconsistent with the location of HCS activity. Improved understanding of biotin biology demands greater knowledge about HCS. Here, we investigated the localisation of HCS and its isoforms. Three variants were observed that differ at the N-terminus. All HCS isoforms were predominantly non-nuclear, consistent with the distribution of biotin protein ligase activity. Unlike the longer constructs, the Met(58) isoform was also detected in the nucleus--a novel observation suggesting shuttling activity between nucleus and cytoplasm. We resolved that the previous controversies in the literature are due to specificity and detection limitations that arise when using partially purified antibodies. 2010. Published by Elsevier Inc. All rights reserved.

  15. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  16. The plastidial folylpolyglutamate synthetase and root apical meristem maintenance

    PubMed Central

    Srivastava, Avinash C; Tang, Yuhong; Díaz de la Garza, Rocío I

    2011-01-01

    Folylpolyglutamate synthetase (FPGS) catalyzes the attachment of glutamate residues to the folate molecule in plants. Three isoforms of FPGS have been identified in Arabidopsis and these are localized in the plastid (AtDFB), mitochondria (AtDFC) and cytosol (AtDFD). We recently determined that mutants in the AtDFB (At5G05980) gene disrupt primary root development in Arabidopsis thaliana seedlings. Transient expression of AtDFB-green fluorescent protein (GFP) fusion under the control of the native AtDFB promoter in Nicotiana tabacum leaf epidermal cells verified the plastid localization of AtDFB. Furthermore, low concentrations of methotrexate (MTX), a compound commonly used as a folate antagonist in plant and mammalian cells induced primary root defects in wild type seedlings that were similar to atdfb. In addition, atdfb seedlings were more sensitive to MTX when compared to wild type. Quantitative (q) RT-PCR showed lower transcript levels of the mitochondrial and cytosolic FPGS in roots of 7-day-old atdfb seedling suggesting feedback regulation of AtDFB on the expression of other FPGS isoforms during early seedling development. The primary root defects of atdfb, which can be traced in part to altered quiescent center (QC) identity, pave the way for future studies that could link cell type specific folate and FPGS isoform requirements to whole organ development. PMID:21502816

  17. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

    PubMed

    Dong, Shi-Hui; Tang, Weixin; Lukk, Tiit; Yu, Yi; Nair, Satish K; van der Donk, Wilfred A

    2015-07-30

    The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

  18. Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNase L

    PubMed Central

    Anderson, Bart R.; Muramatsu, Hiromi; Jha, Babal K.; Silverman, Robert H.; Weissman, Drew; Karikó, Katalin

    2011-01-01

    The interferon-induced enzymes 2′-5′-oligoadenylate synthetase (OAS) and RNase L are key components of innate immunity involved in sensory and effector functions following viral infections. Upon binding target RNA, OAS is activated to produce 2′-5′-linked oligoadenylates (2-5A) that activate RNase L, which then cleaves single-stranded self and non-self RNA. Modified nucleosides that are present in cellular transcripts have been shown to suppress activation of several RNA sensors. Here, we demonstrate that in vitro transcribed, unmodified RNA activates OAS, induces RNase L-mediated ribosomal RNA (rRNA) cleavage and is rapidly cleaved by RNase L. In contrast, RNA containing modified nucleosides activates OAS less efficiently and induces limited rRNA cleavage. Nucleoside modifications also make RNA resistant to cleavage by RNase L. Examining translation in RNase L−/− cells and mice confirmed that RNase L activity reduces translation of unmodified mRNA, which is not observed with modified mRNA. Additionally, mRNA containing the nucleoside modification pseudouridine is translated longer and has an extended half-life. The observation that modified nucleosides in RNA reduce 2-5A pathway activation joins OAS and RNase L to the list of RNA sensors and effectors whose functions are limited when RNA is modified, confirming the role of nucleoside modifications in suppressing immune recognition of RNA. PMID:21813458

  19. Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms

    PubMed Central

    Pateman, J. A.

    1969-01-01

    1. Aspergillus nidulans, Neurospora crassa and Escherichia coli were grown on media containing a range of concentrations of nitrate, or ammonia, or urea, or l-glutamate, or l-glutamine as the sole source of nitrogen and the glutamate dehydrogenate and glutamine synthetase of the cells measured. 2. Aspergillus, Neurospora and Escherichia coli cells, grown on l-glutamate or on high concentrations of ammonia or on high concentrations of urea, possessed low glutamate dehydrogenase activity compared with cells grown on other nitrogen sources. 3. Aspergillus, Neurospora and Escherichia coli cells grown on l-glutamate possessed high glutamine synthetase activity compared with cells grown on other nitrogen sources. 4. The hypothesis is proposed that in Aspergillus, Neurospora and Escherichia coli l-glutamate represses the synthesis of glutamate dehydrogenase and l-glutamine represses the synthesis of glutamine synthetase. 5. A comparison of the glutamine-synthesizing activity and the γ-glutamyltransferase activity of glutamine synthetase in Aspergillus and Neurospora gave no indication that these fungi produce different forms of glutamine synthetase when grown on ammonia or l-glutamate as nitrogen sources. PMID:4901826

  20. Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms.

    PubMed

    Pateman, J A

    1969-12-01

    1. Aspergillus nidulans, Neurospora crassa and Escherichia coli were grown on media containing a range of concentrations of nitrate, or ammonia, or urea, or l-glutamate, or l-glutamine as the sole source of nitrogen and the glutamate dehydrogenate and glutamine synthetase of the cells measured. 2. Aspergillus, Neurospora and Escherichia coli cells, grown on l-glutamate or on high concentrations of ammonia or on high concentrations of urea, possessed low glutamate dehydrogenase activity compared with cells grown on other nitrogen sources. 3. Aspergillus, Neurospora and Escherichia coli cells grown on l-glutamate possessed high glutamine synthetase activity compared with cells grown on other nitrogen sources. 4. The hypothesis is proposed that in Aspergillus, Neurospora and Escherichia colil-glutamate represses the synthesis of glutamate dehydrogenase and l-glutamine represses the synthesis of glutamine synthetase. 5. A comparison of the glutamine-synthesizing activity and the gamma-glutamyltransferase activity of glutamine synthetase in Aspergillus and Neurospora gave no indication that these fungi produce different forms of glutamine synthetase when grown on ammonia or l-glutamate as nitrogen sources.

  1. The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases.

    PubMed

    Chatton, B; Walter, P; Ebel, J P; Lacroute, F; Fasiolo, F

    1988-01-05

    S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

  2. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    SciTech Connect

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A.

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  3. Interdomain and Intermodule Organization in Epimerization Domain Containing Nonribosomal Peptide Synthetases.

    PubMed

    Chen, Wei-Hung; Li, Kunhua; Guntaka, Naga Sandhya; Bruner, Steven D

    2016-08-19

    Nonribosomal peptide synthetases are large, complex multidomain enzymes responsible for the biosynthesis of a wide range of peptidic natural products. Inherent to synthetase chemistry is the thioester templated mechanism that relies on protein/protein interactions and interdomain dynamics. Several questions related to structure and mechanism remain to be addressed, including the incorporation of accessory domains and intermodule interactions. The inclusion of nonproteinogenic d-amino acids into peptide frameworks is a common and important modification for bioactive nonribosomal peptides. Epimerization domains, embedded in nonribosomal peptide synthetases assembly lines, catalyze the l- to d-amino acid conversion. Here we report the structure of the epimerization domain/peptidyl carrier protein didomain construct from the first module of the cyclic peptide antibiotic gramicidin synthetase. Both holo (phosphopantethiene post-translationally modified) and apo structures were determined, each representing catalytically relevant conformations of the two domains. The structures provide insight into domain-domain recognition, substrate delivery during the assembly line process, in addition to the structural organization of homologous condensation domains, canonical players in all synthetase modules.

  4. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

    PubMed Central

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P.; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed. PMID:26270653

  5. Cysteinyl-tRNA synthetase is not essential for viability of the archaeon Methanococcus maripaludis

    PubMed Central

    Stathopoulos, Constantinos; Kim, Wonduck; Li, Tong; Anderson, Iain; Deutsch, Britta; Palioura, Sotiria; Whitman, William; Söll, Dieter

    2001-01-01

    The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell. PMID:11717392

  6. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

    PubMed

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

  7. Glutamate-Dependent Translational Control of Glutamine Synthetase in Bergmann Glia Cells.

    PubMed

    Tiburcio-Félix, Reynaldo; Escalante-López, Miguel; López-Bayghen, Bruno; Martínez, Daniel; Hernández-Kelly, Luisa C; Zinker, Samuel; Hernández-Melchor, Dinorah; López-Bayghen, Esther; Olivares-Bañuelos, Tatiana N; Ortega, Arturo

    2017-09-05

    Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors. A tight regulation of glutamate extracellular levels prevents neuronal overstimulation and cell death, and it is critically involved in glutamate turnover. Glial glutamate transporters are responsible of the majority of the brain glutamate uptake activity. Once internalized, this excitatory amino acid is rapidly metabolized to glutamine via the astrocyte-enriched enzyme glutamine synthetase. A coupling between glutamate uptake and glutamine synthesis and release has been commonly known as the glutamate/glutamine shuttle. Taking advantage of the established model of cultured Bergmann glia cells, in this contribution, we explored the gene expression regulation of glutamine synthetase. A time- and dose-dependent regulation of glutamine synthetase protein and activity levels was found. Moreover, glutamate exposure resulted in the transient shift of glutamine synthetase mRNA from the monosomal to the polysomal fraction. These results demonstrate a novel mode of glutamate-dependent glutamine synthetase regulation and strengthen the notion of an exquisite glia neuronal interaction in glutamatergic synapses.

  8. When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

    PubMed Central

    Roy, Hervé; Becker, Hubert Dominique; Reinbolt, Joseph; Kern, Daniel

    2003-01-01

    Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism. PMID:12874385

  9. Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

    PubMed Central

    Aslanian, A M; Fletcher, B S; Kilberg, M S

    2001-01-01

    Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate. PMID:11415466

  10. Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation.

    PubMed

    Hayano, M; Yang, W S; Corn, C K; Pagano, N C; Stockwell, B R

    2016-02-01

    Ferroptosis is a form of regulated non-apoptotic cell death that has been implicated in several disease contexts. A better understanding of the ferroptotic death mechanism could lead to the development of new therapeutics for degenerative diseases, and a better understanding of how to induce ferroptosis in specific tumor contexts. We performed an unbiased genome-wide siRNA screen to find genetic suppressors of ferroptosis. We determined that loss of CARS, the cysteinyl-tRNA synthetase, suppresses ferroptosis induced by erastin, which inhibits the cystine-glutamate antiporter known as system xc(-). Knockdown of CARS inhibited erastin-induced death by preventing the induction of lipid reactive oxygen species, without altering iron homeostasis. Knockdown of CARS led to the accumulation of cystathionine, a metabolite on the transsulfuration pathway, and upregulated genes associated with serine biosynthesis and transsulfuration. In addition, inhibition of the transsulfuration pathway resensitized cells to erastin, even after CARS knockdown. These studies demonstrate a new mechanism of resistance to ferroptosis and may lead to strategies for inducing and suppressing ferroptosis in diverse contexts.

  11. The U6 snRNA m(6)A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

    PubMed

    Pendleton, Kathryn E; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V; Xie, Yang; Tu, Benjamin P; Conrad, Nicholas K

    2017-05-18

    Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N(6)-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase

    PubMed Central

    Oyola, Samuel O.; Evans, Krystal J.; Smith, Terry K.; Smith, Barbara A.; Hilley, James D.; Mottram, Jeremy C.; Kaye, Paul M.; Smith, Deborah F.

    2012-01-01

    The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism. PMID:23251490

  13. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase

    PubMed Central

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S.; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M. R. K.; Freund, Yvonne R.; DeRisi, Joseph; Cusack, Stephen

    2016-01-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum. Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [14C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS. PMID:27270277

  14. Functional interactions between a glutamine synthetase promoter and MYB proteins.

    PubMed

    Gómez-Maldonado, Josefa; Avila, Concepción; Torre, Fernando; Cañas, Rafael; Cánovas, Francisco M; Campbell, Malcolm M

    2004-08-01

    In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

  15. Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens.

    PubMed

    Gulick, Andrew M

    2017-08-02

    Covering: up to 2017.Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

  16. [2'-5' olygoadenylate synthetase activity in peripheral facial paralysis].

    PubMed

    Nakazato, H; Ikeda, M

    1995-03-01

    Interferons are produced in response to viral infection and play an important part in defense by their antiviral effects. An interferon-induced enzyme, 2'-5' oligoadenylate synthetase (2-5AS) also takes an important part of the system of defense against viral infections, and its activity elevates in nonspecific viral infections. This study was designed to evaluate the usefulness of examining serum 2-5AS activity and peripheral blood WBC 2-5AS (WBC 2-5AS) as diagnostic aids of viral infections that cause facial paralysis. Samples were obtained from 83 patients with Bell's palsy, 20 with Ramsay Hunt syndrome, 74 healthy individuals, and a total of 177 subjects. In 177, we measured serum 2-5AS level in 123 subjects, WBC 2-5AS level in 57, and both in 25. Serum 2-5AS levels in Bell's palsy (60 cases) ranged from 20 to 146 pmol/dl (average: 38.5). The range in Ramsay Hunt syndrome (13) was 20-333 (average: 59.0), and in healthy controls (50), it was 20-128 (average: 41.4). WBC 2-5AS level ranged from 20 to 5900 pmol/dl (average: 733.2) in Bell's palsy (23 cases), from 20-4540 (average: 1371.4) in Ramsay Hunt syndrome (7), and from 20-903 (average: 294.5) in healthy individuals (24). There were no statistically significant differences in serum 2-5AS activities. Otherwise, there was significant difference (p < 0.01) between healthy individuals and Patients with Ramsay Hunt syndrome in WBC 2-5AS activity. In Bell's palsy, 3 cases (13.0%) with markedly high WBC 2-5AS levels existed.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. How does glutamine synthetase activity determine plant tolerance to ammonium?

    PubMed

    Cruz, C; Bio, A F M; Domínguez-Valdivia, M D; Aparicio-Tejo, P M; Lamsfus, C; Martins-Loução, M A

    2006-04-01

    The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants' capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.

  18. Blockade of Glutamine Synthetase Enhances Inflammatory Response in Microglial Cells

    PubMed Central

    Palmieri, Erika M.; Menga, Alessio; Lebrun, Aurore; Hooper, Douglas C.; Butterfield, D. Allan

    2017-01-01

    Abstract Aims: Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia. Results: GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced. Innovation: Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target. Conclusions: Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351–363. PMID:27758118

  19. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2015-10-20

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  20. Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

    Treesearch

    S.C. Minocha; R. Minocha; A. Komamine

    1991-01-01

    Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

  1. Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

    2012-05-08

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  2. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    SciTech Connect

    Schultz, Peter; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2006-08-01

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  3. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    PubMed

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  4. Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

    PubMed Central

    Amaar, Y G; Baillie, D L

    1993-01-01

    In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region. PMID:8414990

  5. Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster

    PubMed Central

    Han, Gye Won; Yang, Xiang-Lei; McMullan, Daniel; Chong, Yeeting E.; Krishna, S. Sri; Rife, Christopher L.; Weekes, Dana; Brittain, Scott M.; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Caruthers, Jonathan; Chiu, Hsiu-Ju; Clayton, Thomas; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Slawomir K.; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Paulsen, Jessica; Reyes, Ron; van den Bedem, Henry; White, Aprilfawn; Wolf, Guenter; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Schimmel, Paul; Wilson, Ian A.

    2010-01-01

    A novel aminoacyl-tRNA synthetase that contains an iron–sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron–sulfur [4Fe–­4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an l-­tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe–4S] cluster-binding motif (C-x 22-C-x 6-C-x 2-C). It is speculated that the iron–sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon. PMID:20944229

  6. SOLUBLE HEPATIC δ-AMINOLEVULINIC ACID SYNTHETASE: END-PRODUCT INHIBITION OF THE PARTIALLY PURIFIED ENZYME*

    PubMed Central

    Scholnick, Perry L.; Hammaker, Lydia E.; Marver, Harvey S.

    1969-01-01

    The present study confirms the existence of hepatic δ-aminolevulinic acid synthetase in the cytosol of the liver, suggests that this enzyme may be in transit to the mitochondria, and defines some of the characteristics of the partially purified enzyme. The substrate and cofactor requirements are similar to those of mitochondrial δ-aminolevulinic acid synthetase. Heme strongly inhibits the partially purified enzyme. A number of proteins that bind heme block this inhibition, which explains previous failures to demonstrate heme inhibition in crude systems. End-product inhibition of δ-aminolevulinic acid synthetase in the mitochondria may play an important role in the regulation of heme biosynthesis in eukaryotic cells. PMID:5257968

  7. Pyrrolysyl-tRNA Synthetase: an ordinary enzyme but an outstanding genetic code expansion tool

    PubMed Central

    Wan, Wei; Tharp, Jeffery M.; Liu, Wenshe R.

    2014-01-01

    The genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs. PMID:24631543

  8. In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803.

    PubMed

    Mérida, A; Candau, P; Florencio, F J

    1991-12-16

    Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.

  9. Archaeal RibL: a new FAD synthetase that is air sensitive.

    PubMed

    Mashhadi, Zahra; Xu, Huimin; Grochowski, Laura L; White, Robert H

    2010-10-12

    FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

  10. Is hydrogen peroxide involved in the benzyl viologen-mediated in-vivo inactivation of rat liver glutamine synthetase?

    PubMed Central

    Muriana, F. J.; Ruiz-Gutierrez, V.; Relimpio, A. M.

    1993-01-01

    After benzyl viologen administration to rats, a decrease in the rat liver glutamine synthetase activity was observed. An increase in the rat liver catalase activity was found concomitantly. In combination with the catalase inhibitor aminotriazole, benzyl viologen again diminished, but markedly, the rat liver glutamine synthetase activity. Moreover, partially purified glutamine synthetase from rat liver underwent rapid inactivation upon aerobic incubation with NAD(P)H and benzyl viologen. This inactivation was prevented by catalase, which suggests that the NAD(P)H/BV2+/O2-dependent system has a role in H2O2 production. Our results suggest that H2O2 is involved in the benzyl viologen-mediated in-vivo inactivation of the rat liver glutamine synthetase. In contrast, benzyl viologen alone or in combination with aminotriazole produced a significant increase of brain glutamine synthetase. PMID:8098954

  11. Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE.

    PubMed

    Jonsson, Anders; Nordlund, Stefan; Teixeira, Pedro Filipe

    2009-10-01

    In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

  12. The binding of tyrosinyl-5'-AMP to tyrosyl-tRNA synthetase (E.coli).

    PubMed Central

    Grosse, F; Krauss, G; Kownatzki, R; Maass, G

    1979-01-01

    The binding between tyrosyl-tRNA synthetase (E.coli) and the alkylanalogue of the aminoacyladenylate, tyrosinyl-5'-AMP, has been investigated by fluorescence titrations and rapid mixing experiments. Tyrosyl-tRNA synthetase has two equivalent and independent binding sites for tyrosinyl-5'-AMP. The intrinsic binding constant is 4 x 10(7)M-1. The binding sites for tRNATyr and tyrosinyl-5'-AMP are independent of each other, the anticooperative mode of tRNA binding being preserved in the presence of tyrosinyl-5-AMP. PMID:377229

  13. Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade.

    PubMed Central

    Varón-Castellanos, R; Havsteen, B H; García-Moreno, M; Valero-Ruiz, E; Molina-Alarcón, M; García-Cánovas, F

    1993-01-01

    A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived. PMID:8104399

  14. A Survey of Glutamine Synthetase Activities in Tissues from Three Classes of Fish.

    DTIC Science & Technology

    1980-09-01

    USA-TR-88-1 NL jj 1-5II!11111112.° MCROCOPY RESOLUTION TEST CHART NATIONAL HIJ|AL) OF STANPARL AL 61 A - USAFA-TR-4O A SURVEY OF GLUTAMINE SYNTHETASE...glutamine synthetase activity is defined as the production of one pmole of y-glutamyl hydroxamate per min at 25°C. Protein was determined by the biuret method...content. P. is listed as at progein per g tissue ( biuret method); nm ± standard deviation.’ Number of specimens examined is listed in parenthesis. 3 body

  15. Correlation of exon 3 β-catenin mutations with glutamine synthetase staining patterns in hepatocellular adenoma and hepatocellular carcinoma.

    PubMed

    Hale, Gillian; Liu, Xinxin; Hu, Junjie; Xu, Zhong; Che, Li; Solomon, David; Tsokos, Christos; Shafizadeh, Nafis; Chen, Xin; Gill, Ryan; Kakar, Sanjay

    2016-11-01

    The current clinical practice is based on the assumption of strong correlation between diffuse glutamine synthetase expression and β-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data and may represent an oversimplification as glutamine synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse glutamine synthetase patterns, and the association between each pattern and β-catenin mutations is not clearly established. This study examines the correlation between glutamine synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified into one of the three patterns: (a) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate-to-strong staining in 50-90% of lesional cells, without a map-like pattern, and (c) patchy: moderate-to-strong staining in <50% of lesional cells (often perivascular), or weak staining irrespective of the extent, and all other staining patterns (including negative cases). Sanger sequencing of CTNNB1 exon 3 was performed in all cases. Of hepatocellular tumors with diffuse glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 β-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 β-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates >50% in both hepatocellular adenoma and hepatocellular

  16. Correlation of Exon 3 β-catenin Mutations with Glutamine Synthetase Staining Patterns in Hepatocellular Adenoma and Hepatocellular Carcinoma

    PubMed Central

    Hale, Gillian; Liu, Xinxin; Hu, Junjie; Xu, Zhong; Che, Li; Solomon, David; Tsokos, Christos; Shafizadeh, Nafis; Chen, Xin; Gill, Ryan; Kakar, Sanjay

    2016-01-01

    The current clinical practice is based on the assumption of strong correlation between diffuse glutamine synthetase expression and β-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data, and may represent an oversimplification as glutamine synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse glutamine synthetase patterns, and the association between each pattern and β-catenin mutations is not clearly established. This study examines the correlation between glutamine synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified into one of three patterns: (a) diffuse homogeneous: moderate to strong cytoplasmic staining in more than 90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate to strong staining in 50–90% of lesional cells, without a map-like pattern, and (c) patchy: moderate to strong staining in <50% of lesional cells (often perivascular), or weak staining irrespective of extent, and all other staining patterns (including negative cases). Sanger sequencing of CTNNB1 exon 3 was performed in all cases. Of hepatocellular tumors with diffuse glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 β-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 β-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates exceeding 50% in both hepatocellular adenoma and

  17. Electromagnetic induction methods

    USDA-ARS?s Scientific Manuscript database

    Electromagnetic induction geophysical methods are finding greater and greater use for agricultural purposes. Electromagnetic induction methods measure the electrical conductivity (or resistivity) for a bulk volume of soil directly beneath the surface. An instrument called a ground conductivity meter...

  18. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases.

    PubMed Central

    Smith, D J; Earl, A J; Turner, G

    1990-01-01

    The nucleotide sequence of the Penicillium chrysogenum Oli13 acvA gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, which performs the first step in penicillin biosynthesis, has been determined. The acvA gene contains an open reading frame of 11,238 bp encoding a protein of 3746 amino acids with a predicted mol. wt of 421,073 dalton. Three domains within the protein of approximately 570 amino acids have between 38% and 43% identity with each other and share similarity with two antibiotic peptide synthetases from Bacillus brevis as well as two other enzymes capable of performing ATP-pyrophosphate exchange reactions. The acvA gene is located close to the pcbC gene encoding isopenicillin N synthetase, the enzyme for the second step of beta-lactam biosynthesis, and is transcribed in the opposite orientation to it. The intergenic region of 1107 bp from which the acvA and pcbC genes are divergently transcribed has also been sequenced. PMID:2118102

  19. A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase.

    PubMed

    Boyarshin, Konstantin S; Priss, Anastasia E; Rayevskiy, Alexsey V; Ilchenko, Mykola M; Dubey, Igor Ya; Kriklivyi, Ivan A; Yaremchuk, Anna D; Tukalo, Michael A

    2017-02-01

    Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA(Pro). The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNA(Pro), which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

  20. The 2-5A/RNase L/RNase L inhibitor (RLI) [correction of (RNI)] pathway regulates mitochondrial mRNAs stability in interferon alpha-treated H9 cells.

    PubMed

    Le Roy, F; Bisbal, C; Silhol, M; Martinand, C; Lebleu, B; Salehzada, T

    2001-12-21

    Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

  1. Teaching Arrangement Inductively.

    ERIC Educational Resources Information Center

    Mendelson, Michael

    1988-01-01

    Argues that teaching arrangement inductively offers an alternative to the standard imitation of business communication text models. Asserts that the inductive method stimulates individual rather than formulaic responses to the problems of organization, and that inductively-trained writers see arrangements as a powerful element in persuasive…

  2. Inductive Reasoning and Writing

    ERIC Educational Resources Information Center

    Rooks, Clay; Boyd, Robert

    2003-01-01

    Induction, properly understood, is not merely a game, nor is it a gimmick, nor is it an artificial way of explaining an element of reasoning. Proper understanding of inductive reasoning--and the various types of reasoning that the authors term inductive--enables the student to evaluate critically other people's writing and enhances the composition…

  3. Induction into Learning Communities

    ERIC Educational Resources Information Center

    Fulton, Kathleen; Yoon, Irene; Lee, Christine

    2005-01-01

    This paper presents the National Commission on Teaching and America's Future (NCTAF) vision for new teacher induction into strong schools that support a career of continuous professional growth. The paper examines data on induction's impact on teacher retention and emerging information on induction's effects on improving student learning, and goes…

  4. Leucyl-tRNA synthetase: double duty in amino acid sensing.

    PubMed

    Durán, Raúl V; Hall, Michael N

    2012-08-01

    The cellular response to amino acids is controlled at the molecular level by TORC1. While many of the elements that participate in TORC1 signaling are known, we still have no clear idea how cells sense amino acids. Two recent studies found that leucyl-tRNA synthetase (LRS) is a leucine sensor for TORC1, in both yeast and mammalian cells.

  5. Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains*

    PubMed Central

    Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

    2015-01-01

    Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1–3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. PMID:26472928

  6. A decrease in S-adenosylmethionine synthetase activity increases the probability of spontaneous sporulation.

    PubMed Central

    Ochi, K; Freese, E

    1982-01-01

    Starting with a relaxed (relA) strain, mutants with reduced activity of adenosine triphosphate:L-methionine S-adenosyl transferase (EC 2.5.1.6; SAM synthetase) were isolated in Bacillus subtilis. One such mutant (gene symbol metE1) had only 3% of the normal SAM synthetase activity but grew almost as well as the parent strain. Another mutant was isolated (gene symbol spdC1) as being able to sporulate continually at a high frequency; it had one-half the normal SAM synthetase activity at 33 degrees C. Both mutants continually and spontaneously entered spore development at a higher frequency than the parent strain in a medium containing excess glucose, ammonium ions, and phosphate. Sporulation was prevented by a high concentration of SAM (1 mM or more) or by the combination of adenosine and methionine (0.5 mM or more each), both of which are precursors of SAM. In contrast to this continual increase in the spore titer, addition of decoyinine, an inhibitor of GMP synthetase, rapidly initiated massive sporulation. Various amino acid analogs also induced sporulation in the relA strain, the methionine analogs ethionine and selenomethionine being most effective. PMID:6811558

  7. Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

    PubMed Central

    Fernandez, M P; Correa, J U; Cabib, E

    1982-01-01

    Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment. Images PMID:6216245

  8. A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

    PubMed Central

    Park, Byung Sun; Yeo, Seung Geun; Jung, Junyang; Jeong, Na Young

    2015-01-01

    Aminoacyl-tRNA synthetases (AminoARSs) are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, AminoARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, AminoARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using AminoARSs-specific primers, we screened mRNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 AminoARSs, we found that phenylalanyl-tRNA synthetase beta chain (FARSB), isoleucyl-tRNA synthetase (IARS) and methionyl-tRNA synthetase (MARS) mRNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment. PMID:26692865

  9. Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

    PubMed Central

    Jetten, M S; Stams, A J; Zehnder, A J

    1989-01-01

    In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii. Images PMID:2571608

  10. Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L. 1

    PubMed Central

    Berger, Michael G.; Sprengart, Michael L.; Kusnan, Misri; Fock, Heinrich P.

    1986-01-01

    Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO2 and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, 15NH4Cl was injected into the central pith of stem sections. The kinetics of 15N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of 15N derived from 15NH4Cl was almost completely inhibited. Injections of amido-15N glutamine demonstrated a potential for 15N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism. PMID:16664820

  11. Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L.

    PubMed

    Berger, M G; Sprengart, M L; Kusnan, M; Fock, H P

    1986-06-01

    Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.

  12. Nucleotide synthetase ribozymes may have emerged first in the RNA world

    PubMed Central

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2007-01-01

    Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

  13. Acetyl-CoA synthetase is a conserved regulator of autophagy and lifespan

    PubMed Central

    Mirzaei, Hamed; Longo, Valter D.

    2014-01-01

    Autophagy is essential for the maintenance of cellular homeostasis during periods of stress. Eisenberg and colleagues (Eisenberg et al., 2014) now describe the central and conserved role for acetyl-CoA synthetase in regulating lifespan in yeast and flies by a mechanism involving autophagy. PMID:24703691

  14. CDC64 Encodes Cytoplasmic Alanyl-tRNA Synthetase, Ala1p, of Saccharomyces cerevisiae

    PubMed Central

    Wrobel, Carolyn; Schmidt, Emmett V.; Polymenis, Michael

    1999-01-01

    The cdc64-1 mutation causes G1 arrest in Saccharomyces cerevisiae corresponding to a type II Start phenotype. We report that CDC64 encodes Ala1p, an alanyl-tRNA synthetase. Thus, cdc64-1 might affect charging of tRNAAla and thereby initiation of cell division. PMID:10601222

  15. Aminoacyl tRNA Synthetase Deficiency Promotes Angiogenesis via the Unfolded Protein Response Pathway

    PubMed Central

    Castranova, Daniel; Davis, Andrew E.; Lo, Brigid D.; Miller, Mayumi F.; Paukstelis, Paul J.; Swift, Matthew R.; Pham, Van N.; Torres-Vázquez, Jesús; Bell, Kameha; Shaw, Kenna M.; Kamei, Makoto; Weinstein, Brant M.

    2016-01-01

    Objective Understanding the mechanisms regulating normal and pathologic angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in two different aminoacyl tRNA synthetases, threonyl tRNA synthetase (tarsy58) or isoleucyl tRNA synthetase (iarsy68), lead to similar increased branching angiogenesis in developing zebrafish. Approach and Results The Unfolded Protein Response (UPR) pathway is activated by aminoacyl tRNA synthetase deficiencies, and we show that UPR genes atf4, atf6, and xbp1, as well as the key pro-angiogenic ligand vascular endothelial growth factor (vegfaa), are all up-regulated in tarsy58 and iarsy68 mutants. Finally, we show that the PERK-ATF4 arm of the UPR pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tarsy58 mutants. Conclusions Our results suggest that endoplasmic reticulum (ER) stress acts as a pro-angiogenic signal via UPR pathway-dependent up-regulation of vegfaa. PMID:26821951

  16. S-Adenosylmethionine synthetase 3 is important for pollen tube growth

    USDA-ARS?s Scientific Manuscript database

    S-Adenosylmethionine is widely used in a variety of biological reactions and participates in the methionine (Met) metabolic pathway. In Arabidopsis (Arabidopsis thaliana), one of the four S-adenosylmethionine synthetase genes, METHIONINE ADENOSYLTRANSFERASE3 (MAT3), is highly expressed in pollen. He...

  17. Ligand co-crystallization of aminoacyl-tRNA synthetases from infectious disease organisms.

    PubMed

    Moen, Spencer O; Edwards, Thomas E; Dranow, David M; Clifton, Matthew C; Sankaran, Banumathi; Van Voorhis, Wesley C; Sharma, Amit; Manoil, Colin; Staker, Bart L; Myler, Peter J; Lorimer, Donald D

    2017-03-16

    Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acid, an essential precursor step to loading of charged tRNAs onto the ribosome and addition of the amino acid to the growing polypeptide chain during protein synthesis. Because of this important biological function, aminoacyl-tRNA synthetases have been the focus of anti-infective drug development efforts and two aaRS inhibitors have been approved as drugs. Several researchers in the scientific community requested aminoacyl-tRNA synthetases to be targeted in the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline. Here we investigate thirty-one aminoacyl-tRNA synthetases from infectious disease organisms by co-crystallization in the presence of their cognate amino acid, ATP, and/or inhibitors. Crystal structures were determined for a CysRS from Borrelia burgdorferi bound to AMP, GluRS from Borrelia burgdorferi and Burkholderia thailandensis bound to glutamic acid, a TrpRS from the eukaryotic pathogen Encephalitozoon cuniculi bound to tryptophan, a HisRS from Burkholderia thailandensis bound to histidine, and a LysRS from Burkholderia thailandensis bound to lysine. Thus, the presence of ligands may promote aaRS crystallization and structure determination. Comparison with homologous structures shows conformational flexibility that appears to be a recurring theme with this enzyme class.

  18. Positive newborn screen in the biochemically normal infant of a mother with treated holocarboxylase synthetase deficiency.

    PubMed

    Nyhan, W L; Willis, M; Barshop, B A; Gangoiti, J

    2009-12-01

    Expanded programmes of newborn screening permit early diagnosis in time to prevent serious complications. These programmes have begun to detect patients who might otherwise remain asymptomatic. An additional confounding variable is the positive screen that results from maternal rather than neonatal disease. This was the case in an infant in whom elevated hydroxyisovalerylcarnitine (C(5)OH) in his newborn screen was the result of placental transfer from his mother, whose holocarboxylase synthetase deficiency was being successfully treated with biotin. The mother had been diagnosed and treated with biotin prenatally. She had no phenotypic feature of holocarboxylase synthetase deficiency, most importantly no episodes ever of acute metabolic acidosis. In the infant a repeat screen was also positive. On day 28 the infant's plasma C(5)OH carnitine was 0.05 mumol/L (normal) and urinary organic acids on day 39 were normal. The mother's excretion of 3-hydroxyisovaleric acid was 109 mmol/mol creatinine. These observations indicate that holocarboxylase synthetase deficiency is one more maternal metabolic disease which may lead to a positive screen in her unaffected newborn infant. They also make the point that holocarboxylase synthetase deficiency in an infant should be detectable in programmes of neonatal screening, which was not clear previously.

  19. Erythrocyte glutathione synthetase in 5-oxoprolinuria: kinetic studies of the mutant enzyme and detection of heterozygotes.

    PubMed

    Larsson, A; Zetterström, R; Hörnell, H; Porath, U

    1976-11-15

    The primary metabolic defect in 5-oxoprolinuria is a generalized deficiency of glutathione synthetase. The activity of this enzyme was determined in cell-free extracts of erythrocytes from patients with 5-oxoprolinuria, their parents and a sibling as well as from normal control individuals. The following activities (pkat/mg of hemoglobin) for glutathione synthetase were obtained: homozygotes mean 0.10 (range 0.07-0.12), heterozygotes mean 3.1 (range 2.8-3.7) and control individuals mean 6.1 (range 5.4-6.7). These results indicate that 5-oxoprolinuria, i.e. the defective gluthione synthetase gene(s), is transmitted by autosomal recessive inheritance. Studies of the kinetics of the low remaining activity of erythrocyte glutathione synthetase in patients with 5-oxoprolinuria failed to reveal defective affinity for glycine, gamma-glutamyl-alpha-aminobutyrate, ATP and Mg2+ ions. Furthermore, the pH optimum, time curves and temperature dependence for the mutant enzyme activity did not significantly differ from the corresponding parameters observed with normal enzyme.

  20. Brain and Liver Glutamine Synthetase of Rana catesbeiana and Rana cancrivora.

    DTIC Science & Technology

    1983-07-01

    glutamine synthetase in the liver is clear for most groups. The lungfishes (Dipnoids) do not retain urea except to avoid ammonia toxicity during...York. 11. Janssens, P.A. and Cohen, P.P. 1968. Nitrogen meta- bolism in the African lungfish . Comp. Biochem. Physiol. 24, 879-886. 9 12. Pickford, G.E

  1. Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

    PubMed Central

    Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

    1999-01-01

    Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

  2. Salmonella typhimurium nit Is nadE: Defective Nitrogen Utilization and Ammonia-Dependent NAD Synthetase

    PubMed Central

    Schneider, Barbara L.; Reitzer, Lawrence J.

    1998-01-01

    S. typhimurium nit mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes. Complementation, enzyme assays, and genetic mapping show that nit is nadE. We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype. PMID:9721319

  3. Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

    PubMed Central

    Lipman, Richard S. A.; Hou, Ya-Ming

    1998-01-01

    Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human. PMID:9811828

  4. A NONSTEADY STATE MODEL FOR THE TIGHT-BINDING INHIBITION OF THYMIDYLATE SYNTHETASE BY 5-FLUOROURACIL

    EPA Science Inventory

    5-Fluorouracil (5_FU) is a widely used chemotherapeutic drug and tratogen that was chosen as a prototypic toxicant to contruct a biologically based dose-resonse (BBDR) model (Setzer et. al., 2001). Part of the BBDR model simulates the inhibition of thymidylate synthetase (TS), a...

  5. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.

  6. Pulsed IR inductive lasers

    NASA Astrophysics Data System (ADS)

    Razhev, A. M.; Churkin, D. S.; Kargapol'tsev, E. S.

    2014-07-01

    Pulsed inductive discharge is a new alternative method of pumping active gas laser media. The work presents results of experimental investigations of near, mid, and far IR inductive gas lasers (H2, HF, and CO2) operating at different transitions of atoms and molecules with different mechanisms of formation of inversion population. The excitation systems of a pulsed inductive cylindrical discharge (pulsed inductively coupled plasma) and pulsed RF inductive discharge in the gases are developed. Various gas mixtures including H2, N2, He, Ne, F2, NF3, and SF6 are used. Characteristics of near IR H2 laser radiation are investigated. Maximal pulse peak power of 7 kW is achieved. The possibility of using a pulsed inductive discharge as a new method of pumping HF laser active medium is demonstrated. The pulsed RF inductive CO2 laser is created and a total efficiency of 17% is achieved.

  7. Dissociation and serenity induction.

    PubMed

    Zoellner, Lori A; Sacks, Matthew B; Foa, Edna B

    2007-09-01

    Dissociation is a common experience during or immediately after a traumatic event; yet, most of the current knowledge regarding dissociation is retrospective in nature. The aim of the present study investigated a non-pharmacological method of dissociative induction with a clinical sample. Participants with PTSD and non-trauma exposed participants were randomly assigned to receive either a dissociative induction, or a serenity induction, based on modified Velten mood induction procedures. Participants receiving the dissociative induction reported higher state-dissociation than those receiving the serenity induction. The PTSD group reported greater state dissociation than the non-trauma exposed group, regardless of induction. State dissociation was related to trait dissociation, PTSD severity, and depression. The present results provide an initial demonstration of the viability for inducing state dissociation in the laboratory with a PTSD sample.

  8. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Δ ura8Δ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  9. The effect of glial glutamine synthetase inhibition on recognition and temporal memories in the rat.

    PubMed

    Kant, Deepika; Tripathi, Shweta; Qureshi, Munazah F; Tripathi, Shweta; Pandey, Swati; Singh, Gunjan; Kumar, Tankesh; Mir, Fayaz A; Jha, Sushil K

    2014-02-07

    The glutamate neurotransmitter is intrinsically involved in learning and memory. Glial glutamine synthetase enzyme synthesizes glutamine, which helps maintain the optimal neuronal glutamate level. However, the role of glutamine synthetase in learning and memory remains unclear. Using associative trace learning task, we investigated the effects of methionine sulfoximine (MSO) (glutamine synthetase inhibitor) on recognition and temporal memories. MSO and vehicle were injected (i.p.) three hours before training in separate groups of male Wistar rats (n=11). Animals were trained to obtain fruit juice after following a set of sequential events. Initially, house-light was presented for 15s followed by 5s trace interval. Thereafter, juice was given for 20s followed by 20s inter-presentation interval. A total of 75 presentations were made over five sessions during the training and testing periods. The average number of head entries to obtain juice per session and during individual phases at different time intervals was accounted as an outcome measure of recognition and temporal memories. The total head entries in MSO and vehicle treated animals were comparable on training and testing days. However, it was 174.90% (p=0.08), 270.61% (p<0.05), 143.20% (p<0.05) more on training day and 270.33% (p<0.05), 157.94% (p<0.05), 170.42% (p<0.05) more on testing day, during the house-light, trace-interval and inter-presentation interval phases in MSO animals. Glutamine synthetase inhibition did not induce recognition memory deficit, while temporal memory was altered, suggesting that glutamine synthetase modulates some aspects of mnemonic processes.

  10. Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum.

    PubMed

    Qiu, Chunhui; Hong, Yang; Cao, Yan; Wang, Fei; Fu, Zhiqiang; Shi, Yaojun; Wei, Meimei; Liu, Shengfa; Lin, Jiaojiao

    2012-12-01

    Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic β-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.μg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

  11. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  12. Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli.

    PubMed

    Thoden, James B; Holden, Hazel M; Firestine, Steven M

    2008-12-16

    N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N(5)-CAIR, MgADP, and P(i). The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N(5)-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P(i) bound in the active site cleft. These structures, determined to 1.6-A resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P(i) complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P(i) rather than ATP. The bound P(i) shifts by approximately 3 A relative to the gamma-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N(5)-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N(5)-CAIR synthetase.

  13. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

    PubMed

    Alegret, M; Ferrando, R; Vázquez, M; Adzet, T; Merlos, M; Laguna, J C

    1994-06-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities. 7. Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man,that are insensitive to peroxisomal proliferation.

  14. Insulin and glucocorticoid dependence of hepatic gamma-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.

    PubMed Central

    Lu, S C; Ge, J L; Kuhlenkamp, J; Kaplowitz, N

    1992-01-01

    We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS. Images PMID:1353765

  15. The tyrosyl-tRNA synthetase like gene located in the tyramine biosynthesis cluster of Enterococcus durans is transcriptionally regulated by tyrosine concentration and extracellular pH

    PubMed Central

    2012-01-01

    Background The tyramine producer Enterococcus durans IPLA655 contains all the necessary genes for tyramine biosynthesis, grouped in the TDC cluster. This cluster includes tyrS, an aminoacyl-tRNA synthetase like gene. Results This work shows that tyrS was maximally transcribed in absence of tyrosine at acidic pH, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Mapping of the tyrS transcriptional start site revealed an unusually long untranslated leader region of 322 bp, which displays the typical features of the T box transcriptional attenuation mechanism. The tyrosine concentration regulation of tyrS was found to be mediated by a transcription antitermination system, whereas the specific induction at acidic pH was regulated at transcription initiation level. Conclusions The expression of the tyrS gene present in the TDC cluster of E. durans is transcriptionally regulated by tyrosine concentration and extracelular pH. The regulation is mediated by both an antitermination system and the promoter itself. PMID:22333391

  16. Sequential inductive learning

    SciTech Connect

    Gratch, J.

    1996-12-31

    This article advocates a new model for inductive learning. Called sequential induction, it helps bridge classical fixed-sample learning techniques (which are efficient but difficult to formally characterize), and worst-case approaches (which provide strong statistical guarantees but are too inefficient for practical use). Learning proceeds as a sequence of decisions which are informed by training data. By analyzing induction at the level of these decisions, and by utilizing the only enough data to make each decision, sequential induction provides statistical guarantees but with substantially less data than worst-case methods require. The sequential inductive model is also useful as a method for determining a sufficient sample size for inductive learning and as such, is relevant to learning problems where the preponderance of data or the cost of gathering data precludes the use of traditional methods.

  17. Nematode asparaginyl-tRNA synthetase resolves intestinal inflammation in mice with T-cell transfer colitis.

    PubMed

    Kron, Michael A; Metwali, Ahmed; Vodanovic-Jankovic, Sanja; Elliott, David

    2013-02-01

    The therapeutic effects of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) have been demonstrated in both animal and human models. However, the inability of individual well-characterized nematode proteins to recreate these beneficial effects has limited the application of component immunotherapy to human disease. The nematodes that cause chronic human lymphatic filariasis, Brugia malayi and Wuchereria bancrofti, are among the parasites that induce immune suppression. Filarial lymphatic pathology has been shown to involve NF-κB pathway-dependent production of vascular endothelial growth factor (VEGF), and stimulation of VEGF expression has also been reported by interleukin 8 (IL-8) via NF-κB pathways. Previously, we have shown that the filarial asparaginyl-tRNA synthetase (rBmAsnRS) interacts with IL-8 receptors using a combination of extracellular loops that differ from those bound by IL-8. To test the hypothesis that rBmAsnRS might induce an anti-inflammatory effect in vivo, we studied the effects of rBmAsnRS in an established murine colitis model using T-cell transfer mice. T-cell transfer colitis mice treated intraperitoneally with 100 μg of rBmAsnRS four times over 2 weeks showed resolution of cellular infiltration in the colonic mucosa, along with induction of a CD8(+) cellular response. In addition, rBmAsnRS induced a rise in IL-10 production from CD3(+) and lipopolysaccharide (LPS)- and cytosine phosphate guanosine (CPG)-stimulated splenic cells. In summary, this work demonstrates a novel anti-inflammatory nematode protein, supports the hygiene hypothesis, and supports continued refinement of alternative immunotherapies for treatment of IBD.

  18. Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases.

    PubMed

    Hara, Ryotaro; Suzuki, Ryohei; Kino, Kuniki

    2015-05-15

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

  19. Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases.

    PubMed Central

    Elseviers, D; Gallagher, P; Hoffman, A; Weinberg, B; Schwartz, I

    1982-01-01

    A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms. Images PMID:6749810

  20. Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases.

    PubMed

    Elseviers, D; Gallagher, P; Hoffman, A; Weinberg, B; Schwartz, I

    1982-10-01

    A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.

  1. Effect of single base substitutions at glycine-870 codon of gramicidin S synthetase 2 gene on proline activation.

    PubMed

    Tokita, K; Hori, K; Kurotsu, T; Kanda, M; Saito, Y

    1993-10-01

    The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.

  2. Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

    DOEpatents

    Anderson, J. Christopher; Wu, Ning; Santoro, Stephen; Schultz, Peter G

    2014-03-11

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

  3. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

    PubMed

    Wray, Lewis V; Fisher, Susan H

    2008-04-01

    The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

  4. A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145.

    PubMed

    Kajiwara, Hitomi; Mine, Toshiki; Miyazaki, Tatsuo; Yamamoto, Takeshi

    2011-01-01

    A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.

  5. The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

    PubMed Central

    McParland, R H; Guevara, J G; Becker, R R; Evans, H J

    1976-01-01

    The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge. Images PLATE 1 PLATE 2 PMID:8035

  6. Proofreading in vivo: Editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli

    SciTech Connect

    Jakubowski, H. )

    1990-06-01

    Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells.

  7. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

    PubMed

    Mirando, Adam C; Fang, Pengfei; Williams, Tamara F; Baldor, Linda C; Howe, Alan K; Ebert, Alicia M; Wilkinson, Barrie; Lounsbury, Karen M; Guo, Min; Francklyn, Christopher S

    2015-08-14

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

  8. Aminoacyl transfer ribonucleic acid synthetases from cell-free extract of Plasmodium berghei.

    PubMed

    Ilan, J; Ilan, J

    1969-05-02

    Aminoacyl transfer ribonucleic acid synthetases for leucine tyrosine, histidine, valine, proline, threonine, and lysine were obtainnned from cell-free extract of Plasmodium berghei. The leucyl-tRNA synthetase cane charge tRNA from liver or Escherichia coli with leucine-c(14), liver tRNA being a better substrate. The amount of aminoacylation increses linerly with respect to the quantity of tRNA added from either source and is dependent on the amount of enzyme added. The rate of aminoacylation is constant for 10 minutes and then decreases. It is enhanced by polyvinylsulfate. One-tenth millimoler pyrimethamine, hydroxystilbamidine, quinacrine, and acriflavine inhibited the formation of C(14)-valyl-tRNA. Species specificity between tRNA and its charging enzyme with respect to the recognition site is discussed.

  9. Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response

    PubMed Central

    Park, Sang Gyu; Kim, Hye Jin; Min, You Hong; Choi, Eung-Chil; Shin, Young Kee; Park, Bum-Joon; Lee, Sang Won; Kim, Sunghoon

    2005-01-01

    Although aminoacyl-tRNA synthetases (ARSs) are essential for protein synthesis, they also function as regulators and signaling molecules in diverse biological processes. Here, we screened 11 different human ARSs to identify the enzyme that is secreted as a signaling molecule. Among them, we found that lysyl-tRNA synthetase (KRS) was secreted from intact human cells, and its secretion was induced by TNF-α. The secreted KRS bound to macrophages and peripheral blood mononuclear cells to enhance the TNF-α production and their migration. The mitogen-activated protein kinases, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, and Gαi were determined to be involved in the signal transduction triggered by KRS. All of these activities demonstrate that human KRS may work as a previously uncharacterized signaling molecule, inducing immune response through the activation of monocyte/macrophages. PMID:15851690

  10. let-65 is cytoplasmic methionyl tRNA synthetase in C. elegans

    PubMed Central

    Alriyami, Maha Z.; Jones, Martin R.; Johnsen, Robert C.; Banerjee, Yajnavalka; Baillie, David L.

    2014-01-01

    Cytoplasmic methionyl tRNA synthetase (MetRS) is one of more than 20 cytoplasmic aminoacyl tRNA synthetase enzymes (ARS). This family of enzymes catalyzes a process fundamental for protein translation. Using a combination of genetic mapping, oligonucleotide array comparative genomic hybridization, and phenotypic correlation, we show that mutations in the essential gene, let-65, reside within the predicted Caenorhabditis elegans homologue of MetRS, which we have named mars-1. We demonstrate that the lethality associated with alleles of let-65 is fully rescued by a transgenic array that spans the mars-1 genomic region. Furthermore, sequence analysis reveals that six let-65 alleles lead to the alteration of highly conserved amino acids. PMID:25606464

  11. Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase.

    PubMed

    Xiao, Han; Peters, Francis B; Yang, Peng-Yu; Reed, Sean; Chittuluru, Johnathan R; Schultz, Peter G

    2014-05-16

    A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUA(Pyl), a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.

  12. Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.

    PubMed Central

    Jakubowski, H

    1990-01-01

    Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells. Images PMID:2191291

  13. Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

    NASA Technical Reports Server (NTRS)

    Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

    1987-01-01

    The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

  14. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  15. Assessing the effects of threonyl-tRNA synthetase on angiogenesis-related responses.

    PubMed

    Mirando, Adam C; Abdi, Khadar; Wo, Peibin; Lounsbury, Karen M

    2017-01-15

    Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays. The theory and discussion include best methods of analysis and quantification along with the advantages and limitations of each type of assay. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  17. Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation.

    PubMed Central

    Francklyn, Christopher; Perona, John J; Puetz, Joern; Hou, Ya-Ming

    2002-01-01

    Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code. PMID:12458790

  18. Structure of Human Phosphopantothenoylcysteine Synthetase at 2.3 Å Resolution

    SciTech Connect

    Manoj, N.; Strauss, E.; Begley, T.P.; Ealick, S.E.

    2010-12-01

    The structure of human phosphopantothenoylcysteine (PPC) synthetase was determined at 2.3 {angstrom} resolution. PPC synthetase is a dimer with identical monomers. Some features of the monomer fold resemble a group of NAD-dependent enzymes, while other features resemble the ribokinase fold. The ATP, phosphopantothenate, and cysteine binding sites were deduced from modeling studies. Highly conserved ATP binding residues include Gly43, Ser61, Gly63, Gly66, Phe230, and Asn258. Highly conserved phosphopantothenate binding residues include Asn59, Ala179, Ala180, and Asp183 from one monomer and Arg55 from the adjacent monomer. The structure predicts a ping pong mechanism with initial formation of an acyladenylate intermediate, followed by release of pyrophosphate and attack by cysteine to form the final products PPC and AMP.

  19. Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

    NASA Technical Reports Server (NTRS)

    Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

    1987-01-01

    The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

  20. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor

    PubMed Central

    Mirando, Adam C.; Fang, Pengfei; Williams, Tamara F.; Baldor, Linda C.; Howe, Alan K.; Ebert, Alicia M.; Wilkinson, Barrie; Lounsbury, Karen M.; Guo, Min; Francklyn, Christopher S.

    2015-01-01

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans. PMID:26271225

  1. Purification, crystallization and data collection of methicillin-resistant Staphylococcus aureus Sar2676, a pantothenate synthetase

    PubMed Central

    Seetharamappa, Jaldappagari; Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal; Overton, Ian M.; van Niekirk, C. A. Johannes; Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F.; Barton, Geoffrey J.; Coote, Peter J.; Naismith, James H.

    2007-01-01

    Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 Å, α = 87.2, β = 81.2, γ = 68.4°. A complete data set has been collected to 2.3 Å resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model. PMID:17554169

  2. Induction: Making the Leap

    ERIC Educational Resources Information Center

    Ling, Lorraine M.

    2009-01-01

    This article provides a critical examination of a variety of approaches to induction focusing especially upon Australia and other Pacific Rim countries. The question of the purposes induction serves for graduate teachers, experienced teachers and education systems is addressed in terms of whether it is a technical exercise which preserves the…

  3. Foundation doctors' induction experiences.

    PubMed

    Miles, Susan; Kellett, Joanne; Leinster, Sam J

    2015-07-24

    It is well established that trainee doctors struggle with the transition from medical school to starting work and feel unprepared for many aspects of their new role. There is evidence that suitable induction experiences improve competence and confidence, but available data indicate that trainee doctors on the UK Foundation Programme are commonly not experiencing useful inductions. The aim of the reported research was to explore trainee doctors' experiences with induction during their first year of the Foundation Programme to identify the most useful characteristics. A questionnaire was designed to explore trainee doctors' experiences with induction at two points during their first Foundation year, during the first and third of three rotations, to enable all induction experiences on offer during the year to be surveyed. Data were collected using an anonymous questionnaire distributed during a teaching session, with an online version available for those trainees not present. Questions gathered information about characteristics of the inductions, usefulness of components of the inductions and what gaps exist. 192 Foundation trainee doctors completed the questionnaire during Rotation 1 and 165 during Rotation 3. The findings indicated that induction experiences at the beginning of the year, including the local Preparation for Professional Practice week, were more useful than those received for later rotations. Longer inductions were more useful than shorter. Departmental inductions were generally only moderately helpful and they missed many important characteristics. Gaps in their inductions identified by many trainees matched those aspects judged to be most useful by those trainees who had experienced these characteristics. Many Foundation trainee doctors are experiencing inadequate inductions, notably at the department level. Trainees are starting rotations in new departments without rudimentary knowledge about their role and responsibilities in that department

  4. Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

    PubMed

    Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

    2012-06-01

    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

  5. Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

    PubMed Central

    Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

    2012-01-01

    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

  6. Quality Control by Isoleucyl-tRNA Synthetase of Bacillus subtilis Is Required for Efficient Sporulation

    PubMed Central

    Kermgard, Elizabeth; Yang, Zhou; Michel, Annika-Marisa; Simari, Rachel; Wong, Jacqueline; Ibba, Michael; Lazazzera, Beth A.

    2017-01-01

    Isoleucyl-tRNA synthetase (IleRS) is an aminoacyl-tRNA synthetase whose essential function is to aminoacylate tRNAIle with isoleucine. Like some other aminoacyl-tRNA synthetases, IleRS can mischarge tRNAIle and correct this misacylation through a separate post-transfer editing function. To explore the biological significance of this editing function, we created a ileS(T233P) mutant of Bacillus subtilis that allows tRNAIle mischarging while retaining wild-type Ile-tRNAIle synthesis activity. As seen in other species defective for aminoacylation quality control, the growth rate of the ileS(T233P) strain was not significantly different from wild-type. When the ileS(T233P) strain was assessed for its ability to promote distinct phenotypes in response to starvation, the ileS(T233P) strain was observed to exhibit a significant defect in formation of environmentally resistant spores. The sporulation defect ranged from 3-fold to 30-fold and was due to a delay in activation of early sporulation genes. The loss of aminoacylation quality control in the ileS(T233P) strain resulted in the inability to compete with a wild-type strain under selective conditions that required sporulation. These data show that the quality control function of IleRS is required in B. subtilis for efficient sporulation and suggests that editing by aminoacyl-tRNA synthetases may be important for survival under starvation/nutrient limitation conditions. PMID:28139725

  7. Glutamine synthetase immunor present in oligodendroglia of regions of the central nervous system

    NASA Technical Reports Server (NTRS)

    D'Amelio, Fernando; Eng, Lawrence F.; Gibbs, Michael A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  8. A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

    PubMed

    Esposito, Marta; Szadocka, Sára; Degiacomi, Giulia; Orena, Beatrice S; Mori, Giorgia; Piano, Valentina; Boldrin, Francesca; Zemanová, Júlia; Huszár, Stanislav; Barros, David; Ekins, Sean; Lelièvre, Joel; Manganelli, Riccardo; Mattevi, Andrea; Pasca, Maria Rosalia; Riccardi, Giovanna; Ballell, Lluis; Mikušová, Katarína; Chiarelli, Laurent R

    2017-06-09

    Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

  9. Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  10. Co-operation between Polymerases and Nucleotide Synthetases in the RNA World

    PubMed Central

    Kim, Ye Eun; Higgs, Paul G.

    2016-01-01

    It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve. PMID:27820829

  11. What is the oligoadenylate synthetases-like protein and does it have therapeutic potential for influenza?

    PubMed

    Alcorn, John F; Sarkar, Saumendra N

    2015-02-01

    Besides its pandemic potential, seasonal influenza infection is associated with an estimated 250,000 to 500,000 deaths worldwide every year. Part of this virulence of influenza virus can be attributed to its ability to evade the host innate immune response. Here, we discuss the possibility of using a recently described mechanism of boosting the innate immunity by oligoadenylate synthetase-like protein, to combat influenza infections.

  12. Glutamine synthetase gene expression and glutamate transporters in C6-glioma cells.

    PubMed

    Baber, Zafeer; Haghighat, Nasrin

    2010-12-01

    Glutamine synthetase (GS) is the major glutamate-forming enzyme of vertebrae and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase in GS activity, and the regulation of this enzyme is the topic of many publications. The amino acid glutamate is the major excitatory neurotransmitter in the brain and mediates normal excitatory synaptic transmission by interaction with postsynaptic receptors. Glutamate also acts as a potent neurotoxin when present at high concentration. Glutamate neurotoxicity plays an important role in the pathophysiology of many neurological disorders, such as Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. In the normal condition, L-glutamate is predominantly taken up, metabolized and recycled by astrocytes through the glutamate transporters (GLAST/GLT1) and glutamine synthetase (GS) catalytic activity. Because of the fundamental role of these glutamate transporters and the glutamine synthetase enzyme in controlling cerebral glutamate level, regulation of GS and studying of the glutamate transporters in glial cells is important. Astrocytes are supportive cells and act as the site of detoxification of glutamate in the brain. However, their isolation from the brain is a tedious, costly and time consuming procedure. On the other hand, the C6-glioma cells are readily available on the market. They are well characterized and have been a useful model for CNS glia in many laboratories. For this study, we used the C6-glioma cell line as a model system. We examined the presence or absence of glial specific glutamate transporters (GLTI and GLAST) in C6-glioma cells, which was done by immunocytochemistry. We also examined glutamine synthetase gene expression in these cells by treatment of the C6-glioma cells with estrogen (17ß estradiol). The findings from this study provide useful information about C6-glioma cells which makes the study of the CNS tremendously inexpensive.

  13. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy.

    PubMed

    Elo, Jenni M; Yadavalli, Srujana S; Euro, Liliya; Isohanni, Pirjo; Götz, Alexandra; Carroll, Christopher J; Valanne, Leena; Alkuraya, Fowzan S; Uusimaa, Johanna; Paetau, Anders; Caruso, Eric M; Pihko, Helena; Ibba, Michael; Tyynismaa, Henna; Suomalainen, Anu

    2012-10-15

    Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.

  14. Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  15. What is the oligoadenylate synthetases-like protein and does it have therapeutic potential for influenza?

    PubMed Central

    Alcorn, John F.; Sarkar, Saumendra N.

    2015-01-01

    Besides its pandemic potential, seasonal influenza infection is associated with an estimated 250,000 to 500,000 deaths worldwide every year. Part of this virulence of influenza virus can be attributed to its ability to evade the host innate immune response. Here we discuss the possibility of using a recently described mechanism of boosting the innate immunity by oligoadenylate synthetase-like protein, to combat influenza infections. PMID:25544107

  16. [Isolation of tyrosyl-tRNA-synthetase from Thermus thermophilus HB-27].

    PubMed

    Iaremchuk, A D; Tukalo, M A; Egorova, S P; Konovalenko, A V; Matsuka, G Kh

    1990-01-01

    A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

  17. Progress in Induction Linacs

    SciTech Connect

    Caporaso, G J

    2000-09-27

    This presentation will be a broad survey of progress in induction technology over the past four years. Much work has been done on accelerators for hydrodynamic test radiography and other applications. Solid-state pulsers have been developed which can provide unprecedented flexibility and precision in pulse format and accelerating voltage for both ion and electron induction machines. Induction linacs can now be built which can operate with MHz repetition rates. Solid-state technology has also made possible the development of fast kickers for precision control of high current beams. New insulator technology has been developed which will improve conventional induction linacs in addition to enabling a new class of high gradient induction linacs.

  18. Principles of Induction Accelerators

    NASA Astrophysics Data System (ADS)

    Briggs*, Richard J.

    The basic concepts involved in induction accelerators are introduced in this chapter. The objective is to provide a foundation for the more detailed coverage of key technology elements and specific applications in the following chapters. A wide variety of induction accelerators are discussed in the following chapters, from the high current linear electron accelerator configurations that have been the main focus of the original developments, to circular configurations like the ion synchrotrons that are the subject of more recent research. The main focus in the present chapter is on the induction module containing the magnetic core that plays the role of a transformer in coupling the pulsed power from the modulator to the charged particle beam. This is the essential common element in all these induction accelerators, and an understanding of the basic processes involved in its operation is the main objective of this chapter. (See [1] for a useful and complementary presentation of the basic principles in induction linacs.)

  19. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    PubMed

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Food safety: Structure and expression of the asparagine synthetase gene family of wheat

    PubMed Central

    Gao, Runhong; Curtis, Tanya Y.; Powers, Stephen J.; Xu, Hongwei; Huang, Jianhua; Halford, Nigel G.

    2016-01-01

    Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat. PMID:27110058

  1. Food safety: Structure and expression of the asparagine synthetase gene family of wheat.

    PubMed

    Gao, Runhong; Curtis, Tanya Y; Powers, Stephen J; Xu, Hongwei; Huang, Jianhua; Halford, Nigel G

    2016-03-01

    Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat.

  2. Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue.

    PubMed

    Levine, R L

    1983-10-10

    Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

  3. Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

    PubMed

    Pai, Chien-Hua; Chiang, Bing-Yu; Ko, Tzu-Ping; Chou, Chia-Cheng; Chong, Cheong-Meng; Yen, Fang-Jiun; Chen, Shoujun; Coward, James K; Wang, Andrew H-J; Lin, Chun-Hung

    2006-12-13

    Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

  4. Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

    PubMed Central

    Ranade, Ranae M.; Zhang, Zhongsheng; Dranow, David M.; Myers, Janette B.; Choi, Ryan; Nakazawa Hewitt, Steve; Edwards, Thomas E.; Davies, Douglas R.; Lorimer, Donald; Boyle, Stephen M.; Barrett, Lynn K.; Buckner, Frederick S.; Fan, Erkang; Van Voorhis, Wesley C.

    2016-01-01

    We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment. PMID:27500735

  5. Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis.

    PubMed

    Ojo, Kayode K; Ranade, Ranae M; Zhang, Zhongsheng; Dranow, David M; Myers, Janette B; Choi, Ryan; Nakazawa Hewitt, Steve; Edwards, Thomas E; Davies, Douglas R; Lorimer, Donald; Boyle, Stephen M; Barrett, Lynn K; Buckner, Frederick S; Fan, Erkang; Van Voorhis, Wesley C

    2016-01-01

    We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

  6. Beneficial consequences of a selective glutamine synthetase inhibitor in oats and legumes

    SciTech Connect

    Langston-Unkefer, P.J.; Knight, T.J.; Sengupta-Gopalan, C.

    1988-01-01

    We report on the effects of administering a unique glutamine synthetase inhibitor to cereals and N/sub 2/-fixing legumes. A bacterium (Pseudomonas syringae pv. tabaci) delivers this inhibitor to provide extended treatment periods; we inoculated the root systems of oat and legume plants with pv. tabaci to provide for delivery of this inhibitor to their root or root/nodule systems. Inoculation of legumes is accompanied by increased plant growth, total plant nitrogen, nodulation, and nitrogen fixation activity. Inoculation of the oats is accompanied by either of two results depending upon the genotype of the oat plant. One result is inhibition of plant growth followed by plant death as consequences of the loss of all of the glutamine synthetase activities in the plant and the subsequent accumulation of ammonia and cessation of nitrate uptake. The second and opposite result is observed in a small population of oats screened from a commercial cultivar and includes increased plant growth and leaf protein. The effects of this inhibitor can be beneficial when applied to appropriate plant material. In an attempt to effectively communicate these findings to the reader, we first introduce the inhibitor (a novel amino acid) and its bacterial delivery systems, the target of the inhibitor (glutamine synthetase-catalyzed ammonia assimilation), and the two different nitrogen economics in the legume and cereal plants used experimentally. The physiological, biochemical, and molecular genetic consequences of the inhibitor action in cereals and legumes, as we presently understand them, are then presented. 18 refs., 4 figs., 3 tabs.,

  7. Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

    PubMed

    Wang, Xiaoyue; Wang, Guanglu; Li, Xinli; Fu, Jing; Chen, Tao; Wang, Zhiwen; Zhao, Xueming

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

  8. Comparison of effects of aspirin and indomethacin on human platelet prostaglandin synthetase.

    PubMed Central

    Crook, D; Collins, A J

    1977-01-01

    Human platelets were incubated in vitro with either aspirin or indomethacin and the prostaglandin synthetase activity of the resultant microsomal fraction from each incubation measured using a radiometric technique. Whereas aspirin produced a dose-related inhibition of the enzyme, indomethacin produced little or no inhibition over the same concentration range (10(-6) mol/l--10(-3) mol/l). Furthermore, administration of aspirin (600 mg) to volunteers produced a highly significant, prolonged inhibition of platelet microsomal prostaglandin synthetase whereas no inhibition was found with indomethacin (50 mg). As indomethacin is considerably more potent than aspirin as an inhibitor of human platelet prostaglandin synthetase in vitro, the results suggest a fundamental difference in the nature of the inhibition produced by each drug, aspirin being an essentially irreversible inhibitor whereas the inhibition produced by indomethacin is reversible. Studies with [3H-acetyl] aspirin have confirmed previous findings (Roth and Majerus, 1975) that aspirin produces an irreversible acetylation of a particulate fraction protein from human platelets. PMID:411427

  9. Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli.

    PubMed Central

    Comer, M M

    1981-01-01

    The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity. PMID:7012115

  10. Cloning and functional characterization of a homoglutathione synthetase from pea nodules.

    PubMed

    Iturbe-Ormaetxe, Iñaki; Heras, Begoña; Matamoros, Manuel A; Ramos, Javier; Moran, Jose F; Becana, Manuel

    2002-05-01

    The thiol tripeptide glutathione (GSH; gammaGlu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; gammaGlu-Cys-betaAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min-1 mg-1 protein with a standard substrate concentration (5 mM beta-alanine) and Km values of 1.9 mM for beta-alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for beta-alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.

  11. Unique domain appended to vertebrate tRNA synthetase is essential for vascular development

    PubMed Central

    Xu, Xiaoling; Shi, Yi; Zhang, Hui-Min; Swindell, Eric C.; Marshall, Alan G.; Guo, Min; Kishi, Shuji; Yang, Xiang-Lei

    2012-01-01

    New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates. PMID:22353712

  12. Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

    PubMed

    Sharma, Arvind; Sharma, Amit

    2015-02-01

    The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

  13. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

    PubMed Central

    Arnez, J G; Harris, D C; Mitschler, A; Rees, B; Francklyn, C S; Moras, D

    1995-01-01

    The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Images PMID:7556055

  14. A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.

    PubMed

    Bembenek, Michael E; Kuhn, Eric; Mallender, William D; Pullen, Lester; Li, Ping; Parsons, Thomas

    2005-10-01

    NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

  15. How induction generators work

    SciTech Connect

    Nailen, R.L.

    1980-06-01

    The operating principles of the induction generator, which is a standard squirrel cage motor in which the shaft is coupled to and driven by an engine or turbine at a rate above its synchronous speed and which, under these conditions, produces electrical power, are described. The advantages of induction generators, e.g., low cost, simplicity, frequency and voltage controlled by the utility system, no synchronizing controls needed, and the advantages of using small induction generators run by wind turbines, small gas turbines and in low head hydro plants are discussed. (LCL)

  16. Direct reading inductance meter

    NASA Technical Reports Server (NTRS)

    Kolby, R. B. (Inventor)

    1977-01-01

    A direct reading inductance meter comprised of a crystal oscillator and an LC tuned oscillator is presented. The oscillators function respectively to generate a reference frequency, f(r), and to generate an initial frequency, f(0), which when mixed produce a difference equal to zero. Upon connecting an inductor of small unknown value in the LC circuit to change its resonant frequency to f(x), a difference frequency (f(r)-f(x)) is produced that is very nearly a linear function of the inductance of the inductor. The difference frequency is measured and displayed on a linear scale in units of inductance.

  17. Properties of inductive reasoning.

    PubMed

    Heit, E

    2000-12-01

    This paper reviews the main psychological phenomena of inductive reasoning, covering 25 years of experimental and model-based research, in particular addressing four questions. First, what makes a case or event generalizable to other cases? Second, what makes a set of cases generalizable? Third, what makes a property or predicate projectable? Fourth, how do psychological models of induction address these results? The key results in inductive reasoning are outlined, and several recent models, including a new Bayesian account, are evaluated with respect to these results. In addition, future directions for experimental and model-based work are proposed.

  18. Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

    PubMed

    Kim, Se-Yu; Park, Soo-Young; Choi, Soo-Keun; Park, Seung-Hwan

    2015-07-01

    The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

  19. Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

    PubMed

    Brown, J R; Doolittle, W F

    1995-03-28

    Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an

  20. Elevated levels of interferon-induced 2'-5' oligoadenylate synthetase in generalized persistent lymphadenopathy and the acquired immunodeficiency syndrome.

    PubMed

    Read, S E; Williams, B R; Coates, R A; Evans, W K; Fanning, M M; Garvey, M B; Shepherd, F A

    1985-09-01

    The levels of the 2'-5' oligoadenylate enzyme synthetase in extracts of peripheral blood mononuclear cells from individuals with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) were measured and compared with synthetase levels in peripheral blood mononuclear cells (PBMs) from healthy heterosexual and homosexual controls. The mean basal synthetase level in heterosexual and homosexual controls was 14 +/- 13 and 12 +/- 9 pmol per hr/10(5) PBMs, respectively. Thirteen individuals with AIDS had a mean basal level of 129 +/- 75 pmol. Serial levels were persistently elevated in six of these individuals over a one- to 10-month period. Twelve of the 13 individuals had antibodies to human T cell lymphotrophic virus-III/lymphadenopathy-associated virus (HTLV-III/LAV). Thirty-three individuals with ARC had a mean basal synthetase level of 68 +/- 84 pmol. Thirty-two of the 33 had antibodies to HTLV-III/LAV. Eleven (33%) have had consistently normal synthetase levels (less than 2 SD above the mean for the homosexual controls, i.e., 30 pmol) over a three- to nine-month follow-up period. Fourteen (42%) had persistently elevated levels over the same period; four (29%) of these developed AIDS during the follow-up period. Eight have had fluctuating levels but have remained clinically well. These studies suggest that persistently elevated synthetase levels in individuals with ARC and antibodies to HTLV-III/LAV indicate progressive virus-induced disease activity. Elevated synthetase levels may be an important prognostic indicator of increased risk of progression to AIDS.

  1. Congenital Visual Impairment and Progressive Microcephaly Due to Lysyl-Transfer Ribonucleic Acid (RNA) Synthetase (KARS) Mutations: The Expanding Phenotype of Aminoacyl-Transfer RNA Synthetase Mutations in Human Disease.

    PubMed

    McMillan, Hugh J; Humphreys, Peter; Smith, Amanda; Schwartzentruber, Jeremy; Chakraborty, Pranesh; Bulman, Dennis E; Beaulieu, Chandree L; Majewski, Jacek; Boycott, Kym M; Geraghty, Michael T

    2015-07-01

    Aminoacyl-transfer ribonucleic acid (RNA) synthetases (ARSs) are a group of enzymes required for the first step of protein translation. Each aminoacyl-transfer RNA synthetase links a specific amino acid to its corresponding transfer RNA component within the cytoplasm, mitochondria, or both. Mutations in ARSs have been linked to a growing number of diseases. Lysyl-transfer RNA synthetase (KARS) links the amino acid lysine to its cognate transfer RNA. We report 2 siblings with severe infantile visual loss, progressive microcephaly, developmental delay, seizures, and abnormal subcortical white matter. Exome sequencing identified mutations within the KARS gene (NM_005548.2):c.1312C>T; p.Arg438Trp and c.1573G>A; p.Glu525Lys occurring within a highly conserved region of the catalytic domain. Our patients' phenotype is remarkably similar to a phenotype recently reported in glutaminyl-transfer RNA synthetase (QARS), another bifunctional ARS gene. This finding expands the phenotypic spectrum associated with mutations in KARS and draws attention to aminoacyl-transfer RNA synthetase as a group of enzymes that are increasingly being implicated in human disease.

  2. Induction melter apparatus

    DOEpatents

    Roach, Jay A [Idaho Falls, ID; Richardson, John G [Idaho Falls, ID; Raivo, Brian D [Idaho Falls, ID; Soelberg, Nicholas R [Idaho Falls, ID

    2008-06-17

    Apparatus and methods of operation are provided for a cold-crucible-induction melter for vitrifying waste wherein a single induction power supply may be used to effect a selected thermal distribution by independently energizing at least two inductors. Also, a bottom drain assembly may be heated by an inductor and may include an electrically resistive heater. The bottom drain assembly may be cooled to solidify molten material passing therethrough to prevent discharge of molten material therefrom. Configurations are provided wherein the induction flux skin depth substantially corresponds with the central longitudinal axis of the crucible. Further, the drain tube may be positioned within the induction flux skin depth in relation to material within the crucible or may be substantially aligned with a direction of flow of molten material within the crucible. An improved head design including four shells forming thermal radiation shields and at least two gas-cooled plenums is also disclosed.

  3. Induction heating coupler

    NASA Technical Reports Server (NTRS)

    Fox, Robert L. (Inventor); Copeland, Carl E. (Inventor); Swaim, Robert J. (Inventor); Coultrip, Robert H. (Inventor); Johnston, David F. (Inventor); Phillips, W. Morris (Inventor); Johnson, Samuel D. (Inventor); Dinkins, James R. (Inventor); Buckley, John D. (Inventor)

    1994-01-01

    An induction heating device includes a handle having a hollow interior and two opposite ends, a wrist connected to one end of the handle, a U-shaped pole piece having two spaced apart ends, a tank circuit including an induction coil wrapped around the pole piece and a capacitor connected to the induction coil, a head connected to the wrist and including a housing for receiving the U-shaped pole piece, the two spaced apart ends of the pole piece extending outwardely beyond the housing, and a power source connected to the tank circuit. When the tank circuit is energized and a susceptor is placed in juxtaposition to the ends of the U-shaped pole piece, the susceptor is heated by induction heating due to magnetic flux passing between the two ends of the pole piece.

  4. Enhancing Induction Coil Reliability

    NASA Astrophysics Data System (ADS)

    Kreter, K.; Goldstein, R.; Yakey, C.; Nemkov, V.

    2014-12-01

    In induction hardening, thermal fatigue is one of the main copper failure modes of induction heat treating coils. There have been papers published that describe this failure mode and others that describe some good design practices. The variables previously identified as the sources of thermal fatigue include radiation from the part surface, frequency, current, concentrator losses, water pressure and coil wall thickness. However, there is very little quantitative data on the factors that influence thermal fatigue in induction coils is available in the public domain. By using finite element analysis software this study analyzes the effect of common design variables of inductor cooling, and quantifies the relative importance of these variables. A comprehensive case study for a single shot induction coil with Fluxtrol A concentrator applied is used for the analysis.

  5. Thermal kinetic inductance detector

    DOEpatents

    Cecil, Thomas; Gades, Lisa; Miceli, Antonio; Quaranta, Orlando

    2016-12-20

    A microcalorimeter for radiation detection that uses superconducting kinetic inductance resonators as the thermometers. The detector is frequency-multiplexed which enables detector systems with a large number of pixels.

  6. Linear induction accelerator

    DOEpatents

    Buttram, M.T.; Ginn, J.W.

    1988-06-21

    A linear induction accelerator includes a plurality of adder cavities arranged in a series and provided in a structure which is evacuated so that a vacuum inductance is provided between each adder cavity and the structure. An energy storage system for the adder cavities includes a pulsed current source and a respective plurality of bipolar converting networks connected thereto. The bipolar high-voltage, high-repetition-rate square pulse train sets and resets the cavities. 4 figs.

  7. Induction of the Proepicardium.

    PubMed

    Maya-Ramos, Lisandro; Cleland, James; Bressan, Michael; Mikawa, Takashi

    2013-09-01

    The proepicardium is a transient extracardiac embryonic tissue that gives rise to the epicardium and a number of coronary vascular cell lineages. This important extracardiac tissue develops through multiple steps of inductive events, from specification of multiple cell lineages to morphogenesis. This article will review our current understanding of inductive events involved in patterning of the proepicardium precursor field, specification of cell types within the proepicardium, and their extension and attachment to the heart.

  8. Induction Heating Systems

    NASA Technical Reports Server (NTRS)

    1991-01-01

    Induction heating technology, a magnetic non-deforming process, was developed by Langley researchers to join plastic and composite components in space. Under NASA license, Inductron Corporation uses the process to produce induction heating systems and equipment for numerous applications. The Torobonder, a portable system, comes with a number of interchangeable heads for aircraft repair. Other developments are the E Heating Head, the Toroid Joining Gun, and the Torobrazer. These products perform bonding applications more quickly, safely and efficiently than previous methods.

  9. Induction launcher design considerations

    NASA Technical Reports Server (NTRS)

    Driga, M. D.; Weldon, W. F.

    1989-01-01

    New concepts in the design of induction accelerators and their power supplies for space and military applications are discussed. Particular attention is given to a piecewise-rising-frequency power supply in which each elementary generator (normal compulsator or rising frequency generator) has a different base frequency. A preliminary design of a coaxial induction accelerator for a hypersonic real gas facility is discussed to illustrate the concepts described.

  10. Doubly fed induction machine

    DOEpatents

    Skeist, S. Merrill; Baker, Richard H.

    2005-10-11

    An electro-mechanical energy conversion system coupled between an energy source and an energy load including an energy converter device having a doubly fed induction machine coupled between the energy source and the energy load to convert the energy from the energy source and to transfer the converted energy to the energy load and an energy transfer multiplexer coupled to the energy converter device to control the flow of power or energy through the doubly fed induction machine.

  11. Induction as Knowledge Integration

    NASA Technical Reports Server (NTRS)

    Smith, Benjamin D.; Rosenbloom, Paul S.

    1996-01-01

    Two key issues for induction algorithms are the accuracy of the learned hypothesis and the computational resources consumed in inducing that hypothesis. One of the most promising ways to improve performance along both dimensions is to make use of additional knowledge. Multi-strategy learning algorithms tackle this problem by employing several strategies for handling different kinds of knowledge in different ways. However, integrating knowledge into an induction algorithm can be difficult when the new knowledge differs significantly from the knowledge the algorithm already uses. In many cases the algorithm must be rewritten. This paper presents Knowledge Integration framework for Induction (KII), a KII, that provides a uniform mechanism for integrating knowledge into induction. In theory, arbitrary knowledge can be integrated with this mechanism, but in practice the knowledge representation language determines both the knowledge that can be integrated, and the costs of integration and induction. By instantiating KII with various set representations, algorithms can be generated at different trade-off points along these dimensions. One instantiation of KII, called RS-KII, is presented that can implement hybrid induction algorithms, depending on which knowledge it utilizes. RS-KII is demonstrated to implement AQ-11, as well as a hybrid algorithm that utilizes a domain theory and noisy examples. Other algorithms are also possible.

  12. The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution.

    PubMed

    Mikami, B; Yoon, H J; Yoshigi, N

    1999-01-22

    The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.

  13. Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin.

    PubMed

    Yonamine, C M; Troncone, L R P; Camillo, M A P

    2004-08-01

    The primary goal of this study was to determine whether Tx2-5, a sodium channel selective toxin obtained from the venom of the spider Phoneutria nigriventer, produced penile erection by means of nitric oxide mechanism. Toxin identity was analyzed by MALDI-TOF, ES-MS and N-terminal amino acid sequencing. Pretreating mice with the non-selective nitric oxide synthase (NOS) inhibitor N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and the selective neuronal-NOS inhibitor 7-Nitroindazole (7-NI) prior to Tx2-5 i.p. (10 microg/25 g mouse) injection challenged the hypothesis above. Controls were injected with the D-isomer or DMSO or saline. Results demonstrated that L-NAME inhibited penile erections in about half the animals treated, while 7-NI completely abolished this effect. Interestingly 7-NI also abolished all the other symptoms of intoxication induced by Tx2-5, including salivation, respiratory distress and death. Tx2-5 killed all the animals of the control group and no one in the 7-NI-treated group. We conclude that (1) intraperitoneal injections of Tx2-5 induce a toxic syndrome that include penile erection, hypersalivation and death by respiratory distress or pulmonary edema; (2) pretreatment with the non-selective NOS inhibitor L-NAME reduces the penile erection and partially protects from the lethal effects of Tx2-5; (3) pretreatment with the nNOS-selective inhibitor 7-NI completely abolishes all the toxic effects of Tx2-5, including penile erection and death suggesting that nNOS is the major player in this intoxication; (4) toxins from other animals that affect sodium channels in the same way as Tx2-5 and induce similar toxic syndromes may have as a major common target, the activation of nitric oxide synthases.

  14. Independent transcription of glutamine synthetase (glnA2) and glutamine synthetase adenylyltransferase (glnE) in Mycobacterium bovis and Mycobacterium tuberculosis.

    PubMed

    Hotter, Grant S; Mouat, Pania; Collins, Desmond M

    2008-09-01

    Mycobacterium bovis and Mycobacterium tuberculosis possess four glutamine synthetase homologues, two of which, glnA1 and glnA2, are required for virulence and are located on the bacterial chromosome on either side of glutamine synthetase adenylyltransferase (glnE). While glnA1 is encoded on the complementary strand, glnA2 is located 48bp upstream from glnE, raising the possibility that glnA2 and glnE may be co-transcribed. However, previous studies in M. bovis and M. tuberculosis have painted a contradictory picture of the (co)transcriptional status of glnA2 and glnE. Given the importance of the genes at the glnA1-glnE-glnA2 locus, we sought to clarify the transcriptional status of glnA2 and glnE in both M. bovis and M. tuberculosis. Reverse transcription-PCR demonstrated that glnA2 and glnE were independently transcribed in all six M. bovis and M. tuberculosis strains examined. Northern analysis of the glnA2 transcript in M. bovis AF2122/97 and M. tuberculosis H37Rv showed that it was monocistronic. These results predicted the presence of a glnE transcriptional start site in the glnA2-glnE intergenic region. An identical start site was confirmed in M. bovis AF2122/97 and M. tuberculosis H37Rv using 5' rapid amplification of cDNA ends. Typical mycobacterial -10 and -35 sequences are associated with this start site.

  15. Effect of post-silking drought on nitrogen partitioning and gene expression patterns of glutamine synthetase and asparagine synthetase in two maize (Zea mays L.) varieties.

    PubMed

    Li, Yajun; Wang, Meiling; Zhang, Fengxia; Xu, Yadong; Chen, Xiaohong; Qin, Xiaoliang; Wen, Xiaoxia

    2016-05-01

    Glutamine synthetase (GS) and asparagine synthetase (AS) are proposed to have important function in plant nitrogen (N) remobilization, but their roles under drought stress are not well defined. In this study, the expression dynamics of GS and AS genes were analyzed in two maize varieties (ZD958 and NH101) in relation to post-silking drought stress induced nitrogen partitioning. ZD958 was a 'stay-green' variety with 5% nitrogen harvest index (NHI) lower than NH101. From silking to maturity, the amount of nitrogen remobilized from ear-leaves in ZD958 was evidently lower than NH101, and post-silking drought stress increased the nitrogen remobilization for both varieties. In ear-leaves, the expression of ZmGln1-3 was enhanced under drought stress. Three AS genes (ZmAS1, ZmAS2 and ZmAS3) were differentially regulated by post-silking drought treatment, of which the expression of ZmAS3 was stimulated at late stage of leaf senescence. In NH101, the expression level of ZmAS3 was markedly higher than that in ZD958. In developing grains, there were no significant differences in expression patterns of GS and AS genes between well water and drought treated plants. Drought stress altered maize N partitioning at the whole-plant level, and the up-regulation of GS and AS genes may contribute to the higher leaf nitrogen remobilization when exposed to drought treatments. Copyright © 2016. Published by Elsevier Masson SAS.

  16. Crystal structure of Pyrococcus horikoshii tryptophanyl-tRNA synthetase and structure-based phylogenetic analysis suggest an archaeal origin of tryptophanyl-tRNA synthetase.

    PubMed

    Dong, Xianchi; Zhou, Minyun; Zhong, Chen; Yang, Bei; Shen, Ning; Ding, Jianping

    2010-03-01

    The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5' AMP (TrpAMP) at 3.0 A resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.

  17. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions.

    PubMed

    Hong, Hye-Jin; Lim, Hui Xuan; Song, Ju Han; Lee, Arim; Kim, Eugene; Cho, Daeho; Cohen, Edward P; Kim, Tae Sung

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

  18. Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

    PubMed Central

    Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

    2014-01-01

    SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

  19. NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803.

    PubMed

    García-Domínguez, M; Reyes, J C; Florencio, F J

    2000-03-01

    Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by direct interaction with two inactivating factors (IF7 and IF17). IF7 and IF17 are homologous polypeptides encoded by the gifA and gifB genes respectively. We investigated the transcriptional regulation of these genes. Expression of both genes is maximum in the presence of ammonium, when GS is inactivated. Nitrogen starvation attenuates the ammonium-mediated induction of gifA and gifB as well as the ammonium-mediated inactivation of GS. Putative binding sites for the transcription factor NtcA were identified at -7.5 and -30.5 bp upstream of gifB and gifA transcription start points respectively. Synechocystis NtcA protein binding to both promoters was demonstrated by gel electrophoresis mobility shift assays. Constitutive high expression levels of both genes were found in a Synechocystis NtcA non-segregated mutant (SNC1), which showed a fourfold reduction in the ntcA expression. These experiments indicate a repressive role for NtcA on the transcription of gifA and gifB genes. Our results demonstrate that NtcA plays a central role in GS regulation in cyanobacteria, stimulating transcription of the glnA gene (GS structural gene) and suppressing transcription of the GS inactivating factor genes gifA and gifB.

  20. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

    PubMed

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Mertens, Haydyn; Svergun, Dmitri; Brieba, Luis G; Grøtli, Morten; Torres-Larios, Alfredo

    2016-07-08

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

  1. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

    PubMed Central

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

    2016-01-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  2. Structural insights into the polyphyletic origins of glycyl tRNA synthetases

    DOE PAGES

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; ...

    2016-05-23

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifexmore » aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. Furthermore, a structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.« less

  3. Targeted disruption of nonribosomal peptide synthetase pes3 augments the virulence of Aspergillus fumigatus.

    PubMed

    O'Hanlon, Karen A; Cairns, Timothy; Stack, Deirdre; Schrettl, Markus; Bignell, Elaine M; Kavanagh, Kevin; Miggin, Sinéad M; O'Keeffe, Grainne; Larsen, Thomas O; Doyle, Sean

    2011-10-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface β-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.

  4. Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases.

    PubMed

    Airas, R Kalervo

    2007-12-01

    The apparent equilibrium constants (K') for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K' values at 1 mM Mg(2+) ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg(2+) concentrations. The values were 2-3 times higher at 20 mM Mg(2+) than at 1 mM Mg(2+), and the dependence was similar in the class I and class II synthetases. The main reason for the Mg(2+) dependence is the existence of PP(i) as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K' on the Mg(2+) concentration is weaker than that caused by PP(i) and AMP. Different bindings of the Mg(2+) ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg(2+) ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg(2+) ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.

  5. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

    SciTech Connect

    Reger,A.; Carney, J.; Gulick, A.

    2007-01-01

    The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

  6. Structural insights into the polyphyletic origins of glycyl tRNA synthetases

    SciTech Connect

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Mertens, Haydyn; Svergun, Dmitri; Brieba, Luis G.; Grøtli, Morten; Torres-Larios, Alfredo

    2016-05-23

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. Furthermore, a structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

  7. Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus).

    PubMed

    Dalessandro, G; Northcote, D H

    1981-01-01

    Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-(14)C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-(14)C]xylose released all the radioactivity as xylose. β-1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4-β xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[(14)C]xylose and tri-O-methyl-[(14)C]xylose, suggesting a 1→4-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent Km value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg(2+) and Mn(2+) ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.

  8. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  9. Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus ▿ †

    PubMed Central

    O'Hanlon, Karen A.; Cairns, Timothy; Stack, Deirdre; Schrettl, Markus; Bignell, Elaine M.; Kavanagh, Kevin; Miggin, Sinéad M.; O'Keeffe, Grainne; Larsen, Thomas O.; Doyle, Sean

    2011-01-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface β-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase. PMID:21746855

  10. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    PubMed

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  11. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

    PubMed Central

    Mérida, A; Candau, P; Florencio, F J

    1991-01-01

    Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium. Images PMID:1676397

  12. Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens)

    Treesearch

    Michelle J. Serapiglia; Rakesh Minocha; Subhash C. Minocha

    2008-01-01

    We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount...

  13. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

    PubMed

    Mérida, A; Candau, P; Florencio, F J

    1991-07-01

    Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.

  14. Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

    PubMed Central

    McArdell, J E; Duffield, M; Atkinson, T

    1989-01-01

    A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates. PMID:2658972

  15. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.

    PubMed

    Jeitner, Thomas M; Cooper, Arthur J L

    2014-12-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.

  16. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase.

    PubMed Central

    Mikkelsen, J; Højrup, P; Rasmussen, M M; Roepstorff, P; Knudsen, J

    1985-01-01

    Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases. PMID:3922356

  17. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    SciTech Connect

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-02-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

  18. Total glutamine synthetase levels in cerebrospinal fluid of Alzheimer's disease patients are unchanged.

    PubMed

    Timmer, Nienke M; Herbert, Megan K; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2015-03-01

    Decreased cerebral protein and activity levels of glutamine synthetase (GS) have been reported for Alzheimer's disease (AD) patients. Using a recently established method, we quantified total GS levels in cerebrospinal fluid (CSF) from AD patients and control subjects. Furthermore, we investigated if total GS levels in CSF could differentiate AD from frontotemperal dementia and dementia with Lewy bodies patients. As we found no significantly altered total GS levels in any of the patient groups compared with control subjects, we conclude that levels of total GS in CSF have no diagnostic value for AD, dementia with Lewy bodies, or frontotemperal dementia.

  19. Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Giardia intestinalis Trophozoites

    PubMed Central

    Ranade, Ranae M.; Zhang, Zhongsheng; Gillespie, J. Robert; Shibata, Sayaka; Verlinde, Christophe L. M. J.; Hol, Wim G. J.; Fan, Erkang

    2015-01-01

    The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs. PMID:26324270

  20. A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase

    PubMed Central

    2015-01-01

    A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively. PMID:26355992

  1. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  2. Characterization of the interaction between lysyl-tRNA synthetase and laminin receptor by NMR.

    PubMed

    Cho, Hye Young; Ul Mushtaq, Ameeq; Lee, Jin Young; Kim, Dae Gyu; Seok, Min Sook; Jang, Minseok; Han, Byung-Woo; Kim, Sunghoon; Jeon, Young Ho

    2014-08-25

    Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS-37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS-LR interaction on the cell surface.

  3. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  4. Effect of Cetyltrimethylammonium Bromide on the Activity of Particulate Starch Synthetase from Potato Tuber

    PubMed Central

    Lavintman, Nelly; Cardini, Carlos E.

    1972-01-01

    The action of some detergents on the incorporation of glucose from uridine diphosphate glucose or adenosine diphosphate glucose into the potato tuber starch grain was studied. It was found that the cationic detergent, cetyltrimethylammonium bromide, produces a rapid binding of both sugar nucleotides to the grain and a great increase in the incorporation of glucose into the polysaccharide. Kinetic constants of starch synthetase are also modified, there being an affinity increase for both sugar nucleotides. Neutral detergents are without effect and anionic detergents are inhibitors. PMID:16658142

  5. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    PubMed Central

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  6. The Role of Pyruvate Dehydrogenase and Acetyl-Coenzyme A Synthetase in Fatty Acid Synthesis in Developing Arabidopsis Seeds1

    PubMed Central

    Ke, Jinshan; Behal, Robert H.; Back, Stephanie L.; Nikolau, Basil J.; Wurtele, Eve Syrkin; Oliver, David J.

    2000-01-01

    Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1α- and ptE1β-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1β mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1β mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds. PMID:10859180

  7. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    SciTech Connect

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  8. Wrapper Induction Software

    SciTech Connect

    2011-08-18

    Wrapper Induction is a software package that allows for unsupervised, semi-supervised, and manual extraction of social media data independent of language or site architecture. A large range of blog formats is available to individuals as means of publishing data to the internet. Blogs are a source of rich information for analysts. With a growing volume of information and blog engines, there is an increased need for automatic or semi-automatic extraction of that data for processing to help deliver results to analysts. Wrapper Induction is designed to automatically or semi-automatically create a template that can be used to harvest blog data from websites. Blogs are in a variety of formats and languages. Wrapper Induction creates a template and extracts blog data in a way that is independent of a specified blog format or language.

  9. Prostaglandin endoperoxide synthetase and the activation of benzo(a)pyrene to reactive metabolites in vivo in guinea pigs

    SciTech Connect

    Garattini, E.; Coccia, P.; Romano, M.; Jiritano, L.; Noseda, A.; Salmona, M.

    1984-11-01

    The role of prostaglandin endoperoxide synthetase in the in vivo activation of benzo(a)pyrene to reactive metabolites capable of interacting irreversibly with cellular macromolecules was studied in guinea pig liver, lung, kidney, spleen, small intestine, colon, and brain. DNA and protein covalent binding experiments were made after systemic administration of acetylsalicylic acid (200 mg/kg) followed by radiolabeled benzo(a)pyrene (4 microgram/kg). Results are compared with a control situation in which the prostaglandin endoperoxide synthetase inhibitor (acetylsalicylic acid) was not administered. No decrease in the level of DNA or protein benzo(a)pyrene-derived covalent binding was observed in any of the tissues studied.

  10. Interaction of aminoacyl-tRNA synthetases and tRNA: positive and negative cooperativity of their active centres.

    PubMed

    Malygin, E G; Zinoviev, V V; Fasiolo, F; Kisselev, L L; Kochkina, L L; Achverdyan, V Z

    1976-07-01

    The influence of tRNA on the kinetics of PP-ATP exchange and aminoacyl-tRNA formation catalysed by leucyl-, phenylalanyl-, and tryptophanyl-tRNA synthetases has been investigated. These enzymes were chosen because they belong to three main classes of quaternary structure alpha1, alpha2beta2 and alpha2, respectively. The present paper shows that the investigated synthetases manifest kinetic cooperativity of the active centres which is negative in the case of AAA formation and positive in the case of leucyl- and tryptophanyl-tRNA synthesis. The obtained data were interpreted with the aid of the trigger model of the enzyme.

  11. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    NASA Astrophysics Data System (ADS)

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  12. Inductive dielectric analyzer

    NASA Astrophysics Data System (ADS)

    Agranovich, Daniel; Polygalov, Eugene; Popov, Ivan; Ben Ishai, Paul; Feldman, Yuri

    2017-03-01

    One of the approaches to bypass the problem of electrode polarization in dielectric measurements is the free electrode method. The advantage of this technique is that, the probing electric field in the material is not supplied by contact electrodes, but rather by electromagnetic induction. We have designed an inductive dielectric analyzer based on a sensor comprising two concentric toroidal coils. In this work, we present an analytic derivation of the relationship between the impedance measured by the sensor and the complex dielectric permittivity of the sample. The obtained relationship was successfully employed to measure the dielectric permittivity and conductivity of various alcohols and aqueous salt solutions.

  13. Induction powered biological radiosonde

    NASA Technical Reports Server (NTRS)

    Fryer, T. B. (Inventor)

    1980-01-01

    An induction powered implanted monitor for epidurally measuring intracranial pressure and telemetering the pressure information to a remote readout is disclosed. The monitor utilizes an inductance-capacitance (L-C) oscillator in which the C comprises a variable capacitance transducer, one electrode of which is a small stiff pressure responsive diaphragm. The oscillator is isolated from a transmitting tank circuit by a buffer circuit and all electric components in the implanted unit except an input and an output coil are shielded by a metal housing.

  14. HIGH GRADIENT INDUCTION ACCELERATOR

    SciTech Connect

    Caporaso, G J; Sampayan, S; Chen, Y; Blackfield, D; Harris, J; Hawkins, S; Holmes, C; Krogh, M; Nelson, S; Nunnally, W; Paul, A; Poole, B; Rhodes, M; Sanders, D; Selenes, K; Sullivan, J; Wang, L; Watson, J

    2007-06-21

    A new type of compact induction accelerator is under development at the Lawrence Livermore National Laboratory that promises to increase the average accelerating gradient by at least an order of magnitude over that of existing induction machines. The machine is based on the use of high gradient vacuum insulators, advanced dielectric materials and switches and is stimulated by the desire for compact flash x-ray radiography sources. Research describing an extreme variant of this technology aimed at proton therapy for cancer will be described. Progress in applying this technology to several applications will be reviewed.

  15. Unobserved "home" induction onto buprenorphine.

    PubMed

    Lee, Joshua D; Vocci, Frank; Fiellin, David A

    2014-01-01

    Unobserved, or "home" buprenorphine induction is common in some clinical practices. Patients take the initial and subsequent doses of buprenorphine after, rather than during, an office visit. This review summarizes the literature on the feasibility and acceptability, safety, effectiveness, and prevalence of unobserved induction. We searched the English language literature for studies describing unobserved buprenorphine induction and associated outcomes. Clinical studies were assessed by strength of design, bias, and internal and external validity. Surveys of provider practices and unobserved induction adoption were reviewed for prevalence data and key findings. We also examined previous review papers and international buprenorphine treatment guidelines. N = 10 clinical studies describing unobserved induction were identified: 1 randomized controlled trial, 3 prospective cohort studies, and 6 retrospective cohort studies. The evidence supports the feasibility of unobserved induction, particularly in office-based primary care practices. Evidence is weak to moderate in support of no differences in adverse event rates between unobserved and observed inductions. There is insufficient or weak evidence in terms of any or no differences in overall effectiveness (treatment retention, medication adherence, illicit opioid abstinence, other drug use). N = 9 provider surveys assessed unobserved induction: observed induction logistics are seen as barriers to buprenorphine prescribing; unobserved induction appears widespread in specific locations. International guidelines reviewed emphasize clinician or pharmacist observed induction (the United States, the United Kingdom, France, Australia); only one (Denmark) explicitly endorses unobserved induction. There is insufficient evidence supporting unobserved induction as more, less, or as effective as observed induction. However, the predominantly observational and naturalistic studies of unobserved induction reviewed, all of which

  16. Vanderwaltozyma polyspora possesses two glycyl-tRNA synthetase genes: one constitutive and one inducible.

    PubMed

    Chien, Chin-I; Chen, Yueh-Lin; Chen, Shun-Jia; Chou, Chi-Mao; Chen, Chin-Yu; Wang, Chien-Chia

    2015-03-01

    Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein synthesis. We herein present evidence that the yeast Vanderwaltozyma polyspora possesses two paralogous glycyl-tRNA synthetase (GlyRS) genes-GRS1 and GRS2. Paradoxically, GRS1 provided functions in both the cytoplasm and mitochondria, while GRS2 was essentially silent under normal growth conditions. Expression of GRS2 could be activated by stresses such as high pH or ethanol and most effectively by high temperature. The expressed GlyRS2 protein was exclusively found in the cytoplasm and more stable under heat-shock conditions (37°C) than under normal growth conditions (30°C) in vivo. In addition, GRS2 effectively rescued the cytoplasmic defect of a Saccharomyces cerevisiae GRS1 knockout strain when expressed from a constitutive promoter. Moreover, the purified GlyRS2 enzyme was fairly active at both 30°C and 37°C in glycylation of yeast tRNA in vitro. However, unexpectedly, the purified GlyRS2 enzyme was practically inactive at temperature above 40°C in vitro. Our study suggests that GRS2 is an inducible gene that acts under stress conditions where GlyRS1 may be insufficient, unavailable, or rendered inactive.

  17. Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations.

    PubMed

    Issoglio, Federico M; Campolo, Nicolas; Zeida, Ari; Grune, Tilman; Radi, Rafael; Estrin, Dario A; Bartesaghi, Silvina

    2016-10-13

    Glutamine synthetase is an important enzyme that catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. In mammals, it plays a key role in preventing excitotoxicity in the brain and detoxifying ammonia in the liver. In plants and bacteria, it is fundamental for nitrogen metabolism, being critical for the survival of the organism. In this work, we show how the use of classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics simulations allowed us to examine the structural properties and dynamics of human glutamine synthetase (HsGS), as well as the reaction mechanisms involved in the catalytic process with atomic level detail. Our results suggest that glutamine formation proceeds through a two-step mechanism that includes a first step in which the γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free energy barrier and a -8 kcal/mol reaction free energy, and then a second rate-limiting step involving the ammonia nucleophilic attack, with a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. A detailed analysis of structural features within each step exposed the relevance of the acid-base equilibrium related to protein residues and substrates in the thermodynamics and kinetics of the reactions. These results provide a comprehensive study of HsGS dynamics and establish the groundwork for further analysis regarding changes in HsGS activity, as occur in natural variants and post-translational modifications.

  18. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.

    PubMed

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-10-19

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  19. Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus.

    PubMed

    Shiyan, Anna; Thompson, Melanie; Köcher, Saskia; Tausendschön, Michaela; Santos, Helena; Hänelt, Inga; Müller, Volker

    2014-01-01

    Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

  20. Structure and transformation of chitin synthetase particles (chitosomes) during microfibril synthesis in vitro.

    PubMed Central

    Bracker, C E; Ruiz-Herrera, J; Bartnicki-Garcia, S

    1976-01-01

    The fine structure of isolated chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamido-deoxyglucosyltransferase; EC 2-4-1-16) particles (chitosomes) from Mucor rouxii and the elaboration of chitin microfibrils were studied by electron microscopy. Chitosomes are spheroidal, but often polymorphic, structures, mostly 40-70 nm in diameter. Their appearance after negative staining varies. Some reveal internal granular structure enclosed by a shell measuring 6-12 nm thick; others do not show internal structure but have a pronounced depression of the external surface. In thin sections, isolated chitosomes appear as microvesicular structures with a tripartite shell 6.5-7.0 nm thick. Morphologically similar structures can be seen in intact cells of M. rouxii. Isolated chitosomes undergo a seemingly irreversible series of transformations when substrate and activators are added. The internal structure changes, and a coiled microfibril (fibroid) appears inside the chitosome. The shell of the chitosome is opened or shed, and an extended microfibril arises from the fibroid particle. During prolonged incubation, the fibroid coils become less common and extended microfibrils appear thicker. We regard the chitosome as the cytoplasmic container and conveyor of chitin synthetase en route to its destination at the cell surface. Isolated chitosomes are well suited for integrated ultrastructural-biochemical studies of microfibril biogenesis in vitro. Images PMID:1070006

  1. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    NASA Technical Reports Server (NTRS)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  2. The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

    PubMed Central

    Mireau, H; Lancelin, D; Small, I D

    1996-01-01

    In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts). To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms. Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs. Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides. A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene. In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme. The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast. We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation. PMID:8672889

  3. The three-dimensional structure of an eukaryotic glutamine synthetase: functional implications of its oligomeric structure.

    PubMed

    Llorca, Oscar; Betti, Marco; González, José M; Valencia, Alfonso; Márquez, Antonio J; Valpuesta, José M

    2006-12-01

    The structure of the prokaryotic glutamine synthetases type I (GS-I), key enzymes in nitrogen metabolism, was determined several years ago by X-ray diffraction, and consists of a double hexameric ring. The structure of the eukaryotic GS from the plant Phaseolus vulgaris (Glutamine synthetase type II; GS-II) has now been determined at low-resolution using electron microscopy and image processing, and consists of an octamer composed of two tetramers placed back-to-back and rotated 90 degrees with respect to each other. The oligomeric structure possesses a twofold symmetry, very suggestive of each tetramer being composed of two dimers. This is reinforced by the fact that dimers are isolated as a stable albeit non-functional species during the purification procedure. Given the fact that the active site of all types of GS is formed by highly conserved residues located in the interface of two interacting monomers, the geometry of the reconstructed tetramer suggests that it only contains two functional active sites, i.e., an active site per dimer. This is supported by biochemical data, which reveal that while the octamer binds eight ATP molecules, it only binds four molecules of the transition state analogue and GS inhibitor methionine-(S)-sulfoximine-P (MetSox-P). All this suggests for the GS-II enzyme an oligomeric structure containing four active sites and four possible regulatory sites, which might point to a complex regulatory behavior.

  4. Structural diversity and protein engineering of the aminoacyl-tRNA synthetases.

    PubMed

    Perona, John J; Hadd, Andrew

    2012-11-06

    Aminoacyl-tRNA synthetases (aaRS) are the enzymes that ensure faithful transmission of genetic information in all living cells, and are central to the developing technologies for expanding the capacity of the translation apparatus to incorporate nonstandard amino acids into proteins in vivo. The 24 known aaRS families are divided into two classes that exhibit functional evolutionary convergence. Each class features an active site domain with a common fold that binds ATP, the amino acid, and the 3'-terminus of tRNA, embellished by idiosyncratic further domains that bind distal portions of the tRNA and enhance specificity. Fidelity in the expression of the genetic code requires that the aaRS be selective for both amino acids and tRNAs, a substantial challenge given the presence of structurally very similar noncognate substrates of both types. Here we comprehensively review central themes concerning the architectures of the protein structures and the remarkable dual-substrate selectivities, with a view toward discerning the most important issues that still substantially limit our capacity for rational protein engineering. A suggested general approach to rational design is presented, which should yield insight into the identities of the protein-RNA motifs at the heart of the genetic code, while also offering a basis for improving the catalytic properties of engineered tRNA synthetases emerging from genetic selections.

  5. Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.

    PubMed

    Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia

    2012-01-01

    Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

  6. 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.

  7. Structural Switch of Lysyl-tRNA Synthetase Between Translation and Transcription

    PubMed Central

    Ofir-Birin, Yifat; Fang, Pengfei; Bennett, Steven P.; Zhang, Hui-Min; Wang, Jing; Rachmin, Inbal; Shapiro, Ryan; Song, Jing; Dagan, Arie; Pozo, Jorge; Kim, Sunghoon; Marshall, Alan G.; Schimmel, Paul; Yang, Xiang-Lei; Nechushtan, Hovav; Razin, Ehud; Guo, Min

    2013-01-01

    SUMMARY Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207-phosphorylation provokes a new conformer of LysRS that inactivates its translational, but activates its transcriptional function. The crystal structure of an MSC sub-complex established that LysRS is held in the MSC by binding to the N-terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap4A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription. PMID:23159739

  8. Saturated Fatty Acid Mutant of Saccharomyces cerevisiae with an Intact Fatty Acid Synthetase

    PubMed Central

    Meyer, Karl H.; Schweizer, Eckhart

    1974-01-01

    A Saccharomyces cerevisiae conditional mutant, LK 181, is described which grows at 37 C only when supplemented with a saturated fatty acid of 12 to 14 carbon atoms chain length. At 22 C, however, no fatty acid supplementation is required for growth. The fatty acid concentration required for optimal growth at 37 C is about four times lower for LK 181 than for fatty acid synthetase-deficient mutants. In contrast to all fatty acid synthetase mutants so far examined, mutant LK 181 cannot grow with palmitic acid. The addition of palmitic, palmitoleic, or oleic acid to the culture medium prevents LK 181 growth at temperatures between 22 and 37 C. In vivo as well as in vitro, cellular de novo fatty acid biosynthesis from acetate is unimpaired in this mutant. It is suggested that endogenously synthesized fatty acids, due to their chain lengths of 16 and more carbon atoms, cannot supplement the mutant LK 181. It is concluded that the exogeneously supplied fatty acids act as allosteric effectors for a mutationally altered cellular protein to restore its biological function at elevated temperatures, rather than as a substitute for endogenously synthesized long-chain fatty acids. PMID:4590462

  9. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    PubMed

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies.

  10. Kinetic abnormalities of carbamyl phosphate synthetase-I in a case of congenital hyperammonaemia.

    PubMed

    Qureshi, I A; Letarte, J; Ouellet, R; Lemieux, B; Cathelineau, L

    1986-01-01

    A sensitive direct colourimetric method has been employed to measure kinetic parameters and pH dependence of carbamyl phosphate synthetase-I, in a liver sample from a 2 1/2-month-old girl, who died from complications of a late-developing congenital hyperammonaemia. The residual activity of carbamyl phosphate synthetase-I was 25%, whereas other urea cycle enzymes were within normal range. Apparent Km for ammonium ion (0.73 mmol/L) was significantly increased (normal range 0.24-0.51). Km for bicarbonate ion was normal, while Km for NAG showed a slight variation from normal. The pH dependence curve of the patient's enzyme was flat, as compared to two controls showing pH optima at 7.8. Radial immunodiffusion (Mancini) of the abnormal enzyme against human enzyme antiserum gave a cross-reacting material of 10-20%. The methodological approach presented can be used to characterize abnormal enzymes in cases of partial deficiency with only 100-200 mg of liver tissue.

  11. Comparison of histidine recognition in human and trypanosomatid histidyl-tRNA synthetases.

    PubMed

    Koh, Cho Yeow; Wetzel, Allan B; de van der Schueren, Will J; Hol, Wim G J

    2014-11-01

    As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS.

  12. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion.

    PubMed

    Szabó, C; Dawson, V L

    1998-07-01

    Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.

  13. Isolation of a Kaurene Synthetase Inhibitor from Castor Bean Seedlings and Cell Suspension Cultures

    PubMed Central

    Gafni, Yedidya; Shechter, Ishaiahu

    1981-01-01

    Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (104-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible. PMID:16661830

  14. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles.

    PubMed

    Piel, Jörn

    2002-10-29

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges.

  15. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    PubMed Central

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-01-01

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition. PMID:27775558

  16. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

    PubMed Central

    Novoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marín-García, Patricia; Azcárate, Isabel G.; Bautista, José M.; Mirando, Adam C.; Francklyn, Christopher S.; Varon, Sònia; Royo, Miriam; Cortés, Alfred; Ribas de Pouplana, Lluís

    2014-01-01

    Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo. PMID:25489076

  17. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    NASA Technical Reports Server (NTRS)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  18. Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

    PubMed

    Suárez, Ramón; Márquez, Judith; Shishkova, Svetlana; Hernández, Georgina

    2003-03-01

    Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

  19. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism

    PubMed Central

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-01-01

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR–DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  20. Glutamine synthetase activity and the expression of three glul paralogues in zebrafish during transport.

    PubMed

    Dhanasiri, Anusha K S; Fernandes, Jorge M O; Kiron, Viswanath

    2012-01-01

    The enzyme glutamine synthetase (GS; glutamate-ammonia ligase, EC 6.3.1.2) plays an important role in the nitrogen metabolism of fish. In this study the GS activity and the corresponding genes were examined to understand how they are regulated in zebrafish in response to hyperammonemic stress during a 72 h simulated transport. Whole body ammonia levels, the activity of the enzyme GS and the mRNA expression of the splice variants of three paralogues of glul, glutamine synthetase gene (glula, glulb and glulc) were examined in brain, liver and kidney of zebrafish. Whole body ammonia reached significantly higher levels by 48 h, while brain showed higher levels as early as 24 h, compared to the values at the start of the transport. The GS activities in brain, liver and kidney were significantly higher at the end of 72 h transport than those at the start. However, only the expression of mRNA of glulb-002 and glulb-003 were significantly upregulated during the simulated transport. In silico analysis of the putative promoter regions of glul paralogues revealed glucocorticoid receptor binding sites. However, glucocorticoid response elements of glulb were not different. The up-regulation of GS enzyme activity and hitherto unreported mRNA expression of glul paralogues during zebrafish transport indicate a physiological response of fish to ammonia.

  1. The Enterococcal Cytolysin Synthetase Coevolves with Substrate for Stereoselective Lanthionine Synthesis.

    PubMed

    Tang, Weixin; Thibodeaux, Gabrielle N; van der Donk, Wilfred A

    2016-09-16

    Stereochemical control is critical in natural product biosynthesis. For ribosomally synthesized and post-translationally modified peptides (RiPPs), the mechanism(s) by which stereoselectivity is achieved is still poorly understood. In this work, we focused on the stereoselective lanthionine synthesis in lanthipeptides, a major class of RiPPs formed by the addition of Cys residues to dehydroalanine (Dha) or dehydrobutyrine (Dhb). Nonenzymatic cyclization of the small subunit of a virulence lanthipeptide, the enterococcal cytolysin, resulted in the native modified peptide as the major product, suggesting that both regioselectivity and stereoselectivity are inherent to the dehydrated peptide sequence. These results support previous computational studies that a Dhx-Dhx-Xxx-Xxx-Cys motif (Dhx = Dha or Dhb; Xxx = any amino acid except Dha, Dhb, and Cys) preferentially cyclizes by attack on the Re face of Dha or Dhb. Characterization of the stereochemistry of the products formed enzymatically with substrate mutants revealed that the lanthionine synthetase actively reinforces Re face attack. These findings support the hypothesis of substrate-controlled selectivity in lanthionine synthesis but also reveal likely coevolution of substrates and lanthionine synthetases to ensure the stereoselective synthesis of lanthipeptides with defined biological activities.

  2. The Enterococcal Cytolysin Synthetase Coevolves with Substrate for Stereoselective Lanthionine Synthesis

    PubMed Central

    Tang, Weixin; Thibodeaux, Gabrielle N.; van der Donk, Wilfred A.

    2017-01-01

    Stereochemical control is critical in natural product biosynthesis. For ribosomally synthesized and post-translationally modified peptides (RiPPs), the mechanism(s) by which stereoselectivity is achieved is still poorly understood. In this work, we focused on the stereoselective lanthionine synthesis in lanthipeptides, a major class of RiPPS formed by addition of Cys residues to dehydroalanine (Dha) or dehydrobutyrine (Dhb). Non-enzymatic cyclization of the small subunit of a virulent lanthipeptide, the enterococcal cytolysin, resulted in the native modified peptide as the major product, suggesting that both regioselectivity and stereoselectivity are inherent to the dehydrated peptide sequence. These results support previous computational studies that a DhxDhxXxxXxxCys motif (Dhx = Dha or Dhb; Xxx = any amino acid except Dha, Dhb, and Cys) preferentially cyclizes by attack on the Re face of Dha or Dhb. Characterization of the stereochemistry of the products formed enzymatically with substrate mutants revealed that the lanthionine synthetase actively reinforces Re face attack. These findings support the hypothesis of substrate-controlled selectivity in lanthionine synthesis but also reveal likely coevolution of substrates and lanthionine synthetases to ensure the stereoselective synthesis of lanthipeptides with defined biological activities. PMID:27348535

  3. Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts.

    PubMed

    Rokov-Plavec, Jasmina; Dulic, Morana; Duchêne, Anne-Marie; Weygand-Durasevic, Ivana

    2008-07-01

    Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.

  4. A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

    PubMed

    Waldron, Ashley L; Cahan, Sara Helms; Franklyn, Christopher S; Ebert, Alicia M

    2017-01-01

    Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

  5. Translocation of the thioesterase domain for the redesign of plipastatin synthetase

    PubMed Central

    Gao, Ling; Liu, Hongxia; Ma, Zhi; Han, Jinzhi; Lu, Zhaoxin; Dai, Chen; Lv, Fengxia; Bie, Xiaomei

    2016-01-01

    Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T–TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system. PMID:28009004

  6. Search for primitive Methanopyrus based on genetic distance between Val- and Ile-tRNA synthetases.

    PubMed

    Yu, Zhiliang; Takai, Ken; Slesarev, Alexei; Xue, Hong; Wong, J Tze-Fei

    2009-10-01

    Since evidence indicates that the Last Universal Common Ancestor (LUCA) was phylogenetically closest to Methanopyrus kandleri among living organisms with elucidated genomes, this study has been directed to a search for the most primitive Methanopyrus lineage. For this purpose, the divergence of valyl-tRNA synthetase (ValRS) and isoleucyl-tRNA synthetase (IleRS) was employed as a measure of primitivity. Comparison of Methanopyrus kandleri and the Methanopyrus isolates GC34 and GC37 from the Pacific Ocean and KOL6, TAG1, TAG11, and SNP6 from the Atlantic Ocean established that the Pacific lineages are more primitive than the Atlantic lineages. Both the groups, however, are younger than environmental genomes from the Kairei Field of Central Indian Ridge in the Indian Ocean. These results showed that different Methanopyrus isolates differ significantly with respect to ValRS-IleRS divergence. On this basis, genomes giving rise to the ValRS and IleRS gene fragments from the Central Indian Ridge represent the most primitive Methanopyrus, phylogenetically the oldest living lineage closest to LUCA.

  7. Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess

    PubMed Central

    Huat, Lim Boon; Garcia, Alfonso Olivos; Ning, Tan Zi; Kin, Wong Weng; Noordin, Rahmah; Azham, Siti Shafiqah Anaqi; Jie, Lee Zhi; Ching, Guee Cher; Chong, Foo Phiaw; Dam, Pim Chau

    2014-01-01

    Objective To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Methods Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. Results A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. Conclusions This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations. PMID:25182945

  8. Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress

    PubMed Central

    Lee, Jin Young; Kim, Dae Gyu; Kim, Byung-Gyu; Yang, Won Suk; Hong, Jeena; Kang, Taehee; Oh, Young Sun; Kim, Kyung Rok; Han, Byung Woo; Hwang, Byung Joon; Kang, Beom Sik; Kang, Mi-Sun; Kim, Myung-Hee; Kwon, Nam Hoon; Kim, Sunghoon

    2014-01-01

    ABSTRACT Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNAMet, leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity. PMID:25097229

  9. One-pot synthesis of class II lanthipeptide bovicin HJ50 via an engineered lanthipeptide synthetase

    PubMed Central

    Wang, Jian; Ge, Xiaoxuan; Zhang, Li; Teng, Kunling; Zhong, Jin

    2016-01-01

    Lanthipeptides are a large class of bacteria-produced, ribosomally-synthesized and post-translationally modified peptides. They are recognized as peptide antibiotics because most of them exhibit potent antimicrobial activities against Gram-positive bacteria especially those that are phylogenetically related to producers. Maturation of class II lanthipeptide like bovicin HJ50 undergoes precursor modification by LanM and a subsequent leader peptide cleavage by LanT. Herein, via co-expression of precursor gene bovA, modification gene bovM and transporter gene bovT in Escherichia coli C43 (DE3), bioactive bovicin HJ50 was successfully produced and secreted. To further achieve in vitro one-pot synthesis of bovicin HJ50, an engineered bovicin HJ50 synthetase BovT150M was obtained by fusing the peptidase domain of BovT (BovT150) to the N-terminus of BovM. BovT150M exhibited dual functions of precursor modification and leader peptide cleavage to release mature bovicin HJ50. Under the guidance of BovA leader peptide, BovT150M exhibited substrate tolerance to modify non-native substrates including suicin and lacticin 481. This work exemplifies the feasibility of enzyme chimera of peptidase domain (LanT150) and modification enzyme (LanM) as a one-pot lanthipeptide synthetase. PMID:27924934

  10. One-pot synthesis of class II lanthipeptide bovicin HJ50 via an engineered lanthipeptide synthetase.

    PubMed

    Wang, Jian; Ge, Xiaoxuan; Zhang, Li; Teng, Kunling; Zhong, Jin

    2016-12-07

    Lanthipeptides are a large class of bacteria-produced, ribosomally-synthesized and post-translationally modified peptides. They are recognized as peptide antibiotics because most of them exhibit potent antimicrobial activities against Gram-positive bacteria especially those that are phylogenetically related to producers. Maturation of class II lanthipeptide like bovicin HJ50 undergoes precursor modification by LanM and a subsequent leader peptide cleavage by LanT. Herein, via co-expression of precursor gene bovA, modification gene bovM and transporter gene bovT in Escherichia coli C43 (DE3), bioactive bovicin HJ50 was successfully produced and secreted. To further achieve in vitro one-pot synthesis of bovicin HJ50, an engineered bovicin HJ50 synthetase BovT150M was obtained by fusing the peptidase domain of BovT (BovT150) to the N-terminus of BovM. BovT150M exhibited dual functions of precursor modification and leader peptide cleavage to release mature bovicin HJ50. Under the guidance of BovA leader peptide, BovT150M exhibited substrate tolerance to modify non-native substrates including suicin and lacticin 481. This work exemplifies the feasibility of enzyme chimera of peptidase domain (LanT150) and modification enzyme (LanM) as a one-pot lanthipeptide synthetase.

  11. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs.

    PubMed

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-04-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

  12. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

    PubMed

    Koryakina, Irina; McArthur, John; Randall, Shan; Draelos, Matthew M; Musiol, Ewa M; Muddiman, David C; Weber, Tilmann; Williams, Gavin J

    2013-01-18

    Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.

  13. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

    PubMed Central

    Steinchen, Wieland; Schuhmacher, Jan S.; Altegoer, Florian; Fage, Christopher D.; Srinivasan, Vasundara; Linne, Uwe; Marahiel, Mohamed A.; Bange, Gert

    2015-01-01

    Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp”] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp—but not ppGpp—positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles. PMID:26460002

  14. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles

    PubMed Central

    Piel, Jörn

    2002-01-01

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges. PMID:12381784

  15. Adenylosuccinate lyase of Bacillus subtilis regulates the activity of the glutamyl-tRNA synthetase.

    PubMed Central

    Gendron, N; Breton, R; Champagne, N; Lapointe, J

    1992-01-01

    In Bacillus subtilis, the glutamyl-tRNA synthetase [L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17] is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability. The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor. The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines. This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B. subtilis. A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient. The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis. Images PMID:1608947

  16. Regulation of Nodule Glutamine Synthetase by CO2 Levels in Bean (Phaseolus vulgaris L.) 1

    PubMed Central

    Ortega, José-Luis; Sánchez, Federico; Soberón, Mario; Flores, Miguel Lara

    1992-01-01

    Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO2 and from day 8 to 17 in high (0.1%) CO2 in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO2 compared with plants grown in 0.02% CO2 and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO2, particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO2 did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the γ polypeptide was significantly reduced in response to high CO2, whereas the β polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper. ImagesFigure 4 PMID:16668681

  17. Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?1

    PubMed Central

    Harrison, Judith; Pou de Crescenzo, Marie-Anne; Sené, Olivier; Hirel, Bertrand

    2003-01-01

    A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T1 progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated. PMID:12970491

  18. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

    PubMed

    Novoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marín-García, Patricia; Azcárate, Isabel G; Bautista, José M; Mirando, Adam C; Francklyn, Christopher S; Varon, Sònia; Royo, Miriam; Cortés, Alfred; Ribas de Pouplana, Lluís

    2014-12-23

    Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

  19. Evidence that tRNA synthetase-directed proton transfer stops mistranslation.

    PubMed

    Waas, William F; Schimmel, Paul

    2007-10-30

    To prevent mistranslation, aminoacyl-tRNA synthetases (AARSs) discriminate against noncognate amino acids and cellular metabolites. Defects in specificity produce statistical proteins which, in mammalian cells, lead to activation of the unfolded protein response and cell death. Because of inherent limitations in amino acid discrimination by a single active site, AARSs evolved a separate domain to clear mischarged amino acids. Although the structure of a widely distributed editing domain for ThrRS and AlaRS is known, the mechanism of amino acid clearance remains elusive. This domain has two motifs that together have four conserved residues in the pocket used to clear serine from mischarged tRNAs. Here, using ThrRS as an example, rapid single-turnover kinetics, mutagenesis, and solvent isotope analysis show that a strictly conserved histidine (between ThrRS and AlaRS) extracts a proton in the chemical step of the editing reaction. Three other conserved residues, and two additional residues in the editing pocket, are not directly implicated in the chemical step. These results are relevant to the previously reported mutagenesis of the homologous editing pocket of alanyl-tRNA synthetase, where even a mild defect in editing causes neurodegeneration in the mouse. Thus, a single proton-transfer event needed to prevent mistranslation can have profound implications for disease.

  20. Training in Inductive Reasoning.

    ERIC Educational Resources Information Center

    Tomic, Welko

    This study investigated the effects of K. J. Klauer's (1989) inductive reasoning training program of teaching children. Effects of training and the range of transfer of the training were assessed. The subjects were 34 third-grade Dutch children of average ability, matched on age, sex, and IQ. Children from the training condition (N=17) received…

  1. Using Continuity Induction

    ERIC Educational Resources Information Center

    Hathaway, Dan

    2011-01-01

    Here is a technique for proving the fundamental theorems of analysis that provides a unified way to pass from local properties to global properties on the real line, just as ordinary induction passes from local implication (if true for "k", the theorem is true for "k" + 1) to a global conclusion in the natural numbers.

  2. Iteration, Not Induction

    ERIC Educational Resources Information Center

    Dobbs, David E.

    2009-01-01

    The main purpose of this note is to present and justify proof via iteration as an intuitive, creative and empowering method that is often available and preferable as an alternative to proofs via either mathematical induction or the well-ordering principle. The method of iteration depends only on the fact that any strictly decreasing sequence of…

  3. Induction technology optimization code

    SciTech Connect

    Caporaso, G.J.; Brooks, A.L.; Kirbie, H.C.

    1992-08-21

    A code has been developed to evaluate relative costs of induction accelerator driver systems for relativistic klystrons. The code incorporates beam generation, transport and pulsed power system constraints to provide an integrated design tool. The code generates an injector/accelerator combination which satisfies the top level requirements and all system constraints once a small number of design choices have been specified (rise time of the injector voltage and aspect ratio of the ferrite induction cores, for example). The code calculates dimensions of accelerator mechanical assemblies and values of all electrical components. Cost factors for machined parts, raw materials and components are applied to yield a total system cost. These costs are then plotted as a function of the two design choices to enable selection of an optimum design based on various criteria. The Induction Technology Optimization Study (ITOS) was undertaken to examine viable combinations of a linear induction accelerator and a relativistic klystron (RK) for high power microwave production. It is proposed, that microwaves from the RK will power a high-gradient accelerator structure for linear collider development. Previous work indicates that the RK will require a nominal 3-MeV, 3-kA electron beam with a 100-ns flat top. The proposed accelerator-RK combination will be a high average power system capable of sustained microwave output at a 300-Hz pulse repetition frequency. The ITOS code models many combinations of injector, accelerator, and pulse power designs that will supply an RK with the beam parameters described above.

  4. Iteration, Not Induction

    ERIC Educational Resources Information Center

    Dobbs, David E.

    2009-01-01

    The main purpose of this note is to present and justify proof via iteration as an intuitive, creative and empowering method that is often available and preferable as an alternative to proofs via either mathematical induction or the well-ordering principle. The method of iteration depends only on the fact that any strictly decreasing sequence of…

  5. Learning and Inductive Inference

    DTIC Science & Technology

    1982-07-01

    is also a paradigm example of induction as discussed by philosophers ( Carnap , 1950). Data compression is of practical value for storage and...IBelser, A. 0. Hlolzman, and A. Kent (Eds.., Eneyclopedia of computer science and technology (Vol. II). New York: Marcel Dekker, 24 51. " Carnap , R. 1950

  6. Educational Inductive Gravimeter

    ERIC Educational Resources Information Center

    Nunn, John

    2014-01-01

    A simple inductive gravimeter constructed from a rigid plastic pipe and insulated copper wire is described. When a magnet is dropped through the vertically mounted pipe it induces small alternating voltages. These small signals are fed to the microphone input of a typical computer and sampled at a typical rate of 44.1 kHz using a custom computer…

  7. Educational Inductive Gravimeter

    ERIC Educational Resources Information Center

    Nunn, John

    2014-01-01

    A simple inductive gravimeter constructed from a rigid plastic pipe and insulated copper wire is described. When a magnet is dropped through the vertically mounted pipe it induces small alternating voltages. These small signals are fed to the microphone input of a typical computer and sampled at a typical rate of 44.1 kHz using a custom computer…

  8. Using Continuity Induction

    ERIC Educational Resources Information Center

    Hathaway, Dan

    2011-01-01

    Here is a technique for proving the fundamental theorems of analysis that provides a unified way to pass from local properties to global properties on the real line, just as ordinary induction passes from local implication (if true for "k", the theorem is true for "k" + 1) to a global conclusion in the natural numbers.

  9. Metallic induction reaction engine

    NASA Astrophysics Data System (ADS)

    Hart, Douglas; Mongeau, Peter P.; Kolm, Henry H.

    1985-11-01

    Metal rings placed close to a pulsed field coil have been accelerated at 200 million gee to 5 km/s in a 2 cm length by Bandoletov in the USSR Bandoletov, 1977. We have studied the basic phenomena and ultimate limitations of the pulsed induction process both theoretically and experimentally to determine its usefulness as a reaction engine. It is possible in principle to accelerate metal rings at high efficiency, and impart sufficient energy to ensure melting and evaporation, so that the reaction mass is ultimately ejected in the form of plasma. In practice the process is limited by electrical, mechanical and thermal failure of the induction coil. Over a hundred shots were fired including several in which 12 gram rings were accelerated to over 700 m/s at efficiencies above 30 percent. This is equivalent to the performance of a high power rifle with a one inch long barrel. An unexpected result of these studies is the discovery that to achieve maximum velocity, the mutual inductance gradient between induction coil and projectile ring in the firing position must be reduced to minimize the initial acceleration. This reduces the back voltage and increases the interaction time, resulting in maximum energy transfer.

  10. Staff Induction: Issues Surrounding Induction into International Schools

    ERIC Educational Resources Information Center

    Stirzaker, Rosalind

    2004-01-01

    This article analyses the literature on staff induction into international schools. It defines what is meant by the term "induction" and identifies the benefits to both individuals and organization if it is done well. It stresses that induction is a process, not an event, and discusses the various stages involved: the initial recruitment, the…

  11. Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

    PubMed Central

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

  12. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    PubMed

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.

  13. The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis.

    PubMed

    Oves-Costales, Daniel; Kadi, Nadia; Challis, Gregory L

    2009-11-21

    Siderophores are high-affinity ferric iron chelators biosynthesised and excreted by most microorganisms that play an important role in iron acquisition. Siderophore-mediated scavenging of ferric iron from hosts contributes significantly to the virulence of pathogenic microbes. As a consequence siderophore biosynthesis is an attractive target for chemotherapeutic intervention. Two main pathways for siderophore biosynthesis exist in microbes. One pathway involves nonribosomal peptide synthetase (NRPS) multienzymes while the other is NRPS-independent. The enzymology of NRPS-mediated siderophore biosynthesis has been extensively studied for more than a decade. In contrast, the enzymology of NRPS-independent siderophore (NIS) biosynthesis was overlooked for almost thirty years since the first genetic characterisation of the NIS biosynthetic pathway to aerobactin. However, the past three years have witnessed an explosion of interest in the enzymology of NIS synthetases, the key enzymes in the assembly of siderophores via the NIS pathway. The biochemical characterisation of ten purified recombinant synthetases has been reported since 2007, along with the first structural characterisation of a synthetase by X-ray crystallography in 2009. In this feature article we summarise the recent progress that has been made in understanding the long-overlooked enzymology of NRPS-independent siderophore biosynthesis, highlight important remaining questions, and suggest likely directions for future research.

  14. Reformation of leucyl-tRNA synthetase complexes in revertants from CHO mutant tsH1.

    PubMed

    Klekamp, M; Pahuski, E; Hampel, A

    1981-11-01

    A direct correlation was found to exist between increased thermolability of leucyl-tRNA synthetase and loss of the high-molecular-weight enzyme complexes in the CHO cell mutant tsH1 and its revertants. This was shown to occur apart from a differential thermostability between the complexes themselves and is supported by Michaelis constant determinations.

  15. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    USDA-ARS?s Scientific Manuscript database

    As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain...

  16. Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

    PubMed Central

    Charlier, J; Sanchez, R

    1987-01-01

    In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product. PMID:3325036

  17. Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

    SciTech Connect

    Guo, Min; Shapiro, Ryan; Schimmel, Paul; Yang, Xiang-Lei

    2010-03-01

    E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization.

  18. Modulation of 2{prime}-5{prime} oligoadenylate synthetase by environmental stress in the marine sponge Geodia cydonium

    SciTech Connect

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G.; Kuusksalu, A.; Kelve, M.

    1997-07-01

    Recently the authors established the presence of relatively high amounts of 2{prime}-5{prime} oligoadenylates (2{prime}-5{prime} A) and 2{prime}-5{prime} oligoadenylate synthetase (2{prime}-5{prime} A synthetase) in the marine sponge Geodia cydonium. Here they determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2{prime}-5{prime} A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2{prime}-5{prime} A content and the activity of 2{prime}-5{prime} A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26 C), cold shock (6 C), pH shock (pH 6), and hypertonic shock and subsequent incubation for 18 h under ambient conditions (16 C). No response was observed after exposure of sponges to an alkaline (pH 10) or hypotonic environment. Similar changes have been found for the expression of heat shock protein HSP70 in G. cydonium. These results show that 2{prime}-5{prime} A in sponges may be useful as a novel biomarker for environmental monitoring.

  19. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix.

    PubMed

    Yoshimura, Yukihiro; Araki, Aya; Maruta, Hitomi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-12-21

    Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

  20. Homoglutathione synthetase and glutathione synthetase in drought-stressed cowpea leaves: expression patterns and accumulation of low-molecular-weight thiols.

    PubMed

    Cruz de Carvalho, Maria H; Brunet, Judicaëlle; Bazin, Jérémie; Kranner, Ilse; d' Arcy-Lameta, Agnès; Zuily-Fodil, Yasmine; Contour-Ansel, Dominique

    2010-04-15

    Glutathione (GSH) is an abundant metabolite and a major antioxidant in plant cells. However, in the Leguminosae, homoglutathione (hGSH) may replace glutathione (GSH) partially or completely. To date, cowpea (Vigna unguiculata) has been considered a non-hGSH-producing species, and no hGSHS cDNA has been isolated. Here we report on the cloning of a full-length cDNA coding for a hGSHS (EC 6.3.2.23) and the cloning of a partial cDNA coding for a putative glutathione synthetase (GSHS; EC 6.3.2.3) in cowpea leaf extracts. These cDNAs possess, respectively, the leucine/proline hGSHS signature and the alanine/alanine GSHS signature at the 3' end. Expression analysis showed a significant up-regulation of hGSHS during progressive drought stress that could be directly related to the drought tolerance of the cowpea cultivar used, while GSHS was mainly constitutively expressed. Nevertheless, quantification of low-molecular-weight thiols confirmed the previous findings that cowpea is essentially a GSH producing plant, as no hGSH was detected in the leaves. These findings raise new questions regarding the function, activity and substrate specificity of the cloned hGSHS cDNA. These questions are discussed. Copyright 2009 Elsevier GmbH. All rights reserved.

  1. Inductive Reasoning: A Training Approach

    ERIC Educational Resources Information Center

    Klauer, Karl Josef; Phye, Gary D.

    2008-01-01

    Researchers have examined inductive reasoning to identify different cognitive processes when participants deal with inductive problems. This article presents a prescriptive theory of inductive reasoning that identifies cognitive processing using a procedural strategy for making comparisons. It is hypothesized that training in the use of the…

  2. Inductive Reasoning: A Training Approach

    ERIC Educational Resources Information Center

    Klauer, Karl Josef; Phye, Gary D.

    2008-01-01

    Researchers have examined inductive reasoning to identify different cognitive processes when participants deal with inductive problems. This article presents a prescriptive theory of inductive reasoning that identifies cognitive processing using a procedural strategy for making comparisons. It is hypothesized that training in the use of the…

  3. Induction plasma tube

    DOEpatents

    Hull, Donald E.

    1984-01-01

    An induction plasma tube having a segmented, fluid-cooled internal radiation shield is disclosed. The individual segments are thick in cross-section such that the shield occupies a substantial fraction of the internal volume of the plasma enclosure, resulting in improved performance and higher sustainable plasma temperatures. The individual segments of the shield are preferably cooled by means of a counterflow fluid cooling system wherein each segment includes a central bore and a fluid supply tube extending into the bore. The counterflow cooling system results in improved cooling of the individual segments and also permits use of relatively larger shield segments which permit improved electromagnetic coupling between the induction coil and a plasma located inside the shield. Four embodiments of the invention, each having particular advantages, are disclosed.

  4. Pulsed inductive HF laser

    NASA Astrophysics Data System (ADS)

    Razhev, A. M.; Churkin, D. S.; Kargapol'tsev, E. S.; Demchuk, S. V.

    2016-03-01

    We report the results of experimentally investigated dependences of temporal, spectral and spatial characteristics of an inductive HF-laser generation on the pump conditions. Gas mixtures H2 - F2(NF3 or SF66) and He(Ne) - H2 - F2(NF3 or SF6) were used as active media. The FWHM pulse duration reached 0.42 μs. This value corresponded to a pulsed power of 45 kW. For the first time, the emission spectrum of an inductive HF laser was investigated, which consisted of seven groups of bands with centres around the wavelengths of 2732, 2736, 2739, 2835, 2837, 2893 and 2913 nm. The cross section profile of the laser beam was a ring with a diameter of about 20 mm and width of about 5 mm. Parameters of laser operation in the repetitively pulsed regime were sufficiently stable. The amplitude instability of light pulses was no greater than 5% - 6%.

  5. Inductive Supervised Quantum Learning

    NASA Astrophysics Data System (ADS)

    Monràs, Alex; Sentís, Gael; Wittek, Peter

    2017-05-01

    In supervised learning, an inductive learning algorithm extracts general rules from observed training instances, then the rules are applied to test instances. We show that this splitting of training and application arises naturally, in the classical setting, from a simple independence requirement with a physical interpretation of being nonsignaling. Thus, two seemingly different definitions of inductive learning happen to coincide. This follows from the properties of classical information that break down in the quantum setup. We prove a quantum de Finetti theorem for quantum channels, which shows that in the quantum case, the equivalence holds in the asymptotic setting, that is, for large numbers of test instances. This reveals a natural analogy between classical learning protocols and their quantum counterparts, justifying a similar treatment, and allowing us to inquire about standard elements in computational learning theory, such as structural risk minimization and sample complexity.

  6. Inductive Supervised Quantum Learning.

    PubMed

    Monràs, Alex; Sentís, Gael; Wittek, Peter

    2017-05-12

    In supervised learning, an inductive learning algorithm extracts general rules from observed training instances, then the rules are applied to test instances. We show that this splitting of training and application arises naturally, in the classical setting, from a simple independence requirement with a physical interpretation of being nonsignaling. Thus, two seemingly different definitions of inductive learning happen to coincide. This follows from the properties of classical information that break down in the quantum setup. We prove a quantum de Finetti theorem for quantum channels, which shows that in the quantum case, the equivalence holds in the asymptotic setting, that is, for large numbers of test instances. This reveals a natural analogy between classical learning protocols and their quantum counterparts, justifying a similar treatment, and allowing us to inquire about standard elements in computational learning theory, such as structural risk minimization and sample complexity.

  7. Induction plasma tube

    DOEpatents

    Hull, D.E.

    1982-07-02

    An induction plasma tube having a segmented, fluid-cooled internal radiation shield is disclosed. The individual segments are thick in cross-section such that the shield occupies a substantial fraction of the internal volume of the plasma enclosure, resulting in improved performance and higher sustainable plasma temperatures. The individual segments of the shield are preferably cooled by means of a counterflow fluid cooling system wherein each segment includes a central bore and a fluid supply tube extending into the bore. The counterflow cooling system results in improved cooling of the individual segments and also permits use of relatively larger shield segments which permit improved electromagnetic coupling between the induction coil and a plasma located inside the shield. Four embodiments of the invention, each having particular advantages, are disclosed.

  8. Induction motor control

    NASA Astrophysics Data System (ADS)

    Hansen, Irving G.

    Electromechanical actuators developed to date have commonly ultilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilized induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high-frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high-frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  9. Induction motor control

    NASA Technical Reports Server (NTRS)

    Hansen, Irving G.

    1990-01-01

    Electromechanical actuators developed to date have commonly ultilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilized induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high-frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high-frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  10. Induction motor control

    NASA Technical Reports Server (NTRS)

    Hansen, Irving G.

    1990-01-01

    Electromechanical actuators developed to date have commonly utilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilizes induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  11. Induction motor control

    NASA Technical Reports Server (NTRS)

    Hansen, Irving G.

    1990-01-01

    Electromechanical actuators developed to date have commonly ultilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilized induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high-frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high-frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  12. Inductive Position Sensor

    NASA Technical Reports Server (NTRS)

    Youngquist, Robert C. (Inventor); Simmons, Stephen M. (Inventor)

    2015-01-01

    An inductive position sensor uses three independent inductors inductively coupled by a common medium such as air. First and second inductors are separated by a fixed distance with the first inductor's axial core and second inductor's axial core maintained parallel to one another. A third inductor is disposed between the first and second inductors with the third inductor's axial core being maintained parallel to those of the first and second inductors. The combination of the first and second inductors are configured for relative movement with the third inductor's axial core remaining parallel to those of the first and second inductors as distance changes from the third inductor to each of the first inductor and second inductor. An oscillating current can be supplied to at least one of the three inductors, while voltage induced in at least one of the three inductors not supplied with the oscillating current is measured.

  13. An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones.

    PubMed

    Cornejo Maciel, Fabiana; Maloberti, Paula; Neuman, Isabel; Cano, Florencia; Castilla, Rocío; Castillo, Fernanda; Paz, Cristina; Podestá, Ernesto J

    2005-06-01

    We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary

  14. Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

    PubMed Central

    Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2003-01-01

    The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

  15. Glucocorticoids induce glutamine synthetase in folliculostellate cells of rat pituitary glands in vivo and in vitro

    PubMed Central

    SHIRASAWA, NOBUYUKI; YAMANOUCHI, HIROSHI

    1999-01-01

    Glutamine synthetase (GS) is a glucocorticoid-inducible enzyme that has a key role for glutamate metabolism in the central and peripheral nervous system. In this study GS activity was measured and the amount of immunoreactive GS (ir-GS) cells in the rat anterior pituitary gland was quantified as a function of age. In addition, the effects of GS inhibitors, glucocorticoid administration, and adrenalectomy on GS activity were examined. Some of the ir-GS cells were also immunoreactive for S100 protein (ir-S100) which is a known marker for folliculostellate cells (FS) in the anterior pituitary. FS cells expressing GS were first detected in 3-d-old rats, and this cell population, expressed as the immunostained cell area divided by a standard unit area, increased as a function of age. The percentages of FS cells also expressing GS were 0.2, 6.4, 25 and 74% at 3 d, 30 d, 60 d and 2 y of age, respectively. GS enzyme activity also increased in parallel with the increase of ir-GS cell population maturation. The subcutaneous injection of methionine sulphoximine, a GS and γ-glutamylcysteine synthetase inhibitor, reduced pituitary GS activity by 83%, but increased the population of ir-GS cells 3.5-fold in 30-d-old rats. Buthionine sulphoximine, a specific inhibitor of γ-glutamylcysteine synthetase, had little effect on GS activity or the ir-GS cell population. Neither methionine sulphoximine nor buthionine sulphoximine changed the population of ir-S100 protein cells (FS cells). Dexamethasone and hydrocortisone increased the population of ir-GS cells by 3.1 and 4.2-fold, respectively, within 12 h after administration. A significant increase of GS activity due to the injection of glucocorticoids was observed in the anterior pituitary, but not in the brain, retina or liver of immature rats. Adrenalectomy did not cause decrease of pituitary GS activity, and dexamethasone administration increased GS activity in both adrenalectomised and intact rats. In the monolayer culture of

  16. Structural Analysis of the Active Site Geometry of N[superscript 5]-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli

    SciTech Connect

    Thoden, James B.; Holden, Hazel M.; Firestine, Steven M.

    2009-09-11

    N{sub 5}-Carboxyaminoimidazole ribonucleotide synthetase (N{sub 5}-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N{sub 5}-CAIR, MgADP, and P{sub i}. The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N{sub 5}-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P{sub i} bound in the active site cleft. These structures, determined to 1.6-{angstrom} resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P{sub i} complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P{sub i} rather than ATP. The bound P{sub i} shifts by approximately 3 {angstrom} relative to the {gamma}-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N{sub 5}-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N{sub 5}-CAIR synthetase.

  17. Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation.

    PubMed

    Euro, Liliya; Konovalova, Svetlana; Asin-Cayuela, Jorge; Tulinius, Már; Griffin, Helen; Horvath, Rita; Taylor, Robert W; Chinnery, Patrick F; Schara, Ulrike; Thorburn, David R; Suomalainen, Anu; Chihade, Joseph; Tyynismaa, Henna

    2015-01-01

    The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

  18. High Gradient Induction Cell

    SciTech Connect

    Caporaso, G J

    2004-11-29

    A concept being developed for high current electron beams may have application to HEDP and is described here. It involves the use of planar Blumlein stacks placed inside an induction cell. The output end of the Blumlein stack is applied across a high gradient insulator (HGI). These insulators have been used successfully in the presence of kilo Ampere-level electron beam currents for tens of nanoseconds at gradients of 20 MV/meter.

  19. Inductively commutated coilguns

    SciTech Connect

    Mongeau, P.P. )

    1991-01-01

    In this paper the concept and relevance of power factor is presented in regards to high performance launchers. As the scale of launchers grows and as efforts to improve efficiency continue power factor considerations will become crucial in engineering design and ultimate launcher performance limits. The use of motion induced commutation to improve the power factor are discussed. Various approaches to inductive commutation are presented, including: the brush-commutated 9 MJ Coilgun, the solid state-switched coilgun and the quenchgun.

  20. Diagnostics for induction accelerators

    SciTech Connect

    Fessenden, T.J.

    1996-04-01

    The induction accelerator was conceived by N. C. Christofilos and first realized as the Astron accelerator that operated at LLNL from the early 1960`s to the end of 1975. This accelerator generated electron beams at energies near 6 MeV with typical currents of 600 Amperes in 400 ns pulses. The Advanced Test Accelerator (ATA) built at Livermore`s Site 300 produced 10,000 Ampere beams with pulse widths of 70 ns at energies approaching 50 MeV. Several other electron and ion induction accelerators have been fabricated at LLNL and LBNL. This paper reviews the principal diagnostics developed through efforts by scientists at both laboratories for measuring the current, position, energy, and emittance of beams generated by these high current, short pulse accelerators. Many of these diagnostics are closely related to those developed for other accelerators. However, the very fast and intense current pulses often require special diagnostic techniques and considerations. The physics and design of the more unique diagnostics developed for electron induction accelerators are presented and discussed in detail.