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Sample records for acetyl-coa synthetase acs

  1. Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii.

    PubMed

    Rodrigues, Fernando; Zeeman, Anne-Marie; Cardoso, Helena; Sousa, Maria João; Steensma, H Yde; Côrte-Real, Manuela; Leão, Cecília

    2004-03-01

    A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.

  2. The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol.

    PubMed

    Kretzschmar, U; Schobert, M; Görisch, H

    2001-10-01

    Pseudomonas aeruginosa ATCC 17933 uses a pyrroloquinoline quinone-dependent ethanol oxidation system. Two mutants of P. aeruginosa, unable to grow on ethanol and showing no acetyl-CoA synthetase (ACS) activity under standard test conditions, were complemented by cosmid pTB3018. Subcloning led to the isolation of a gene which encodes a protein with high similarity to acetyl-CoA synthetases. Interruption of the putative acsA gene by a kanamycin-resistance cassette resulted in a mutant also unable to grow on ethanol and with very low residual acetyl-CoA-forming activity. Complementation by the wild-type allele of the acsA gene restored growth and led to the expression of ACS activity in excess of that of wild-type cells. In wild-type P. aeruginosa, ACS activity was induced upon growth on ethanol, 2,3-butanediol, malonate and acetate. The wild-type and mutants defective in ACS activity showed an active acetate kinase (ACK) under the growth conditions used; however, phosphotransacetylase (PTA) could not be detected. The data indicate that P. aeruginosa requires active acsA gene product for growth on ethanol.

  3. Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea

    PubMed Central

    Barnhart, Elliott P.; McClure, Marcella A.; Johnson, Kiki; Cleveland, Sean; Hunt, Kristopher A.; Fields, Matthew W.

    2015-01-01

    Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life. PMID:26235787

  4. Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea

    DOE PAGES

    Barnhart, Elliott P.; McClure, Marcella A.; Johnson, Kiki; ...

    2015-08-03

    Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- andmore » ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. Lastly, these results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.« less

  5. Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea

    SciTech Connect

    Barnhart, Elliott P.; McClure, Marcella A.; Johnson, Kiki; Cleveland, Sean; Hunt, Kristopher A.; Fields, Matthew W.

    2015-08-03

    Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. Lastly, these results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

  6. 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.

  7. 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

  8. 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.

  9. [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.

  10. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. 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.

  12. 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 ...

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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 ...

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis.

    PubMed

    Kozak, Barbara U; van Rossum, Harmen M; Benjamin, Kirsten R; Wu, Liang; Daran, Jean-Marc G; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acsacs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h(-1) were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20 h(-1)) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor.

  5. 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

  6. Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway.

    PubMed

    Ohkuni, Aya; Ohno, Yusuke; Kihara, Akio

    2013-12-13

    Sphingosine 1-phosphate (S1P) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in S1P metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where S1P metabolism takes place. We previously proposed the entire S1P metabolic pathway from results obtained using yeast cells, i.e., S1P is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as S1P is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that S1P is metabolized by a common pathway in eukaryotes.

  7. 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.].

  8. 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

  9. 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

  10. Giardia fatty acyl-CoA synthetases as potential drug targets

    PubMed Central

    Guo, Fengguang; Ortega-Pierres, Guadalupe; Argüello-García, Raúl; Zhang, Haili; Zhu, Guan

    2015-01-01

    Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids (FA) de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate FA scavenged from the host. ACS is an essential enzyme because FA need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference toward palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis–Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 μM, Ki = 0.18 μM for GiACS1, and IC50 = 2.28 μM, Ki = 0.23 μM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 μM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite. PMID:26257723

  11. Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

    PubMed Central

    Tullius, M. V.; Vann, W. F.; Gibson, B. W.

    1999-01-01

    Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP. PMID:10091669

  12. 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.

  13. 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.

  14. Stability Characteristics of "Aerobic" Acetyl-CoA Synthetase of Yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Klein, Harold P.

    1976-01-01

    During the purification of the "aerobic" acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae, strain LK2Gl2, it was noted that stronge at 4 C resulted in the loss of enzyme activity within 24 hr. Similar losses were observed during column chromatography. Addition of boiled extracts from either aerobic or anerobic cells completely prevents this. The stabilizing factor (SF) in these extracts is non-dialyzable and organic in nature. SF is excluded on G-25 and G-50 Sephadex columns and is slightly retarded on G-75 columns. On G-100 columns, SF elutes as a peak exactly coincident with that of cytochrome c, indicating a molecular weight of 13,000. SF activity was not destroyed by Pronase treatment, was adsorbed onto Norite, and absorbed in the UV with a single maximum at 260 nm. The action of SF could be replaced by a number of nucleotides. At 0.01 M, the order of effectiveness was: ATP>ADP>AMP>GTP>CTP>/=UTP>XTP. Even at 2 x 10(exp -4) M, ATP and ADP, but not AMP, cyclic AMP, adenosine or adenine, were effective in stabilizing this ACS. The mechanism of stabilization by ATP and AMP appears to be the same, since AMP competitively inhibited the ACS with respect to ATP in in vitro assays, while ADP gave a mixed type of inhibition, thus indicating a different mechanism. ACS from nonaerobic cells is also unstable in the absence of SF but, unlike aerobic ACS, is not affected by ATP or other nucleotides.

  15. 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

  16. 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

  17. 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

  18. Characterization and expression of AMP-forming Acetyl-CoA Synthetase from Dunaliella tertiolecta and its response to nitrogen starvation stress

    PubMed Central

    Liang, Ming-Hua; Qv, Xiao-Ying; Jin, Hong-Hao; Jiang, Jian-Guo

    2016-01-01

    AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1–A10) and three signature motifs (I–III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrapTM HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition. PMID:27025661

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis

    PubMed Central

    Mayer, Florian; Küper, Ulf; Meyer, Carolin; Daxer, Stefanie; Müller, Volker; Rachel, Reinhard

    2012-01-01

    Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO2 fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H2:sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO2 fixation. PMID:22247508

  8. Reversible Nε-Lysine Acetylation Regulates the Activity of Acyl-CoA Synthetases Involved in Anaerobic Benzoate Catabolism in Rhodopseudomonas palustris

    PubMed Central

    Crosby, Heidi A.; Heiniger, Erin K.; Harwood, Caroline S.; Escalante-Semerena, Jorge C.

    2010-01-01

    Rhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available in aquatic environments rich in plant-derived lignin. Benzoate degradation is regulated at the transcriptional level in R. palustris in response to anoxia and the presence of benzoate and/or benzoyl-CoA (Bz-CoA). Here, we report evidence that anaerobic benzoate catabolism in this bacterium is also regulated at the posttranslational level. In this pathway, benzoate is activated to Bz-CoA by the AMP-forming Bz-CoA synthetase (BadA) enzyme. Mass spectrometry and mutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation of Lys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4-hydroxybenzoyl-CoA (HbaA) and cyclohexanecarboxyl-CoA (AliA) synthetases were also reversibly acetylated. We identified one acetyltransferase that modified BadA, Hba, and AliA in vitro. The acetyltransferase enzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica sv Typhimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl-CoA (Ac-CoA) synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivate BadAAc. One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin-type NAD+-dependent deacetylase (O-acetyl-ADP-ribose-forming); the other deacetylase is LdaA (encoded by ldaA, for lysine deacetylase A; formerly rpa0954), an acetate-forming protein deacetylase. LdaA reactivated HbaAc and AliAAc in vitro. PMID:20345662

  9. 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.

  10. 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.

  11. ACS: ALMA Common Software

    NASA Astrophysics Data System (ADS)

    Chiozzi, Gianluca; Šekoranja, Matej

    2013-02-01

    ALMA Common Software (ACS) provides a software infrastructure common to all ALMA partners and consists of a documented collection of common patterns and components which implement those patterns. The heart of ACS is based on a distributed Component-Container model, with ACS Components implemented as CORBA objects in any of the supported programming languages. ACS provides common CORBA-based services such as logging, error and alarm management, configuration database and lifecycle management. Although designed for ALMA, ACS can and is being used in other control systems and distributed software projects, since it implements proven design patterns using state of the art, reliable technology. It also allows, through the use of well-known standard constructs and components, that other team members whom are not authors of ACS easily understand the architecture of software modules, making maintenance affordable even on a very large project.

  12. 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

  13. Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus

    DOE PAGES

    Scott, Joseph W.; Poole, Farris L.; Adams, Michael W. W.

    2014-01-01

    Tmore » he hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 β 2 structures but the genome contains genes encoding three additional α -subunits.he ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes.he α -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids.he β -subunit determined preference for adenine or guanine nucleotides.he GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes.ranscriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of thehermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.« less

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Microfabricated AC impedance sensor

    DOEpatents

    Krulevitch, Peter; Ackler, Harold D.; Becker, Frederick; Boser, Bernhard E.; Eldredge, Adam B.; Fuller, Christopher K.; Gascoyne, Peter R. C.; Hamilton, Julie K.; Swierkowski, Stefan P.; Wang, Xiao-Bo

    2002-01-01

    A microfabricated instrument for detecting and identifying cells and other particles based on alternating current (AC) impedance measurements. The microfabricated AC impedance sensor includes two critical elements: 1) a microfluidic chip, preferably of glass substrates, having at least one microchannel therein and with electrodes patterned on both substrates, and 2) electrical circuits that connect to the electrodes on the microfluidic chip and detect signals associated with particles traveling down the microchannels. These circuits enable multiple AC impedance measurements of individual particles at high throughput rates with sufficient resolution to identify different particle and cell types as appropriate for environmental detection and clinical diagnostic applications.

  2. 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.

  3. 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.

  4. AC magnetohydrodynamic microfluidic switch

    SciTech Connect

    Lemoff, A V; Lee, A P

    2000-03-02

    A microfluidic switch has been demonstrated using an AC Magnetohydrodynamic (MHD) pumping mechanism in which the Lorentz force is used to pump an electrolytic solution. By integrating two AC MHD pumps into different arms of a Y-shaped fluidic circuit, flow can be switched between the two arms. This type of switch can be used to produce complex fluidic routing, which may have multiple applications in {micro}TAS.

  5. Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Klein, Harold P.

    1976-01-01

    A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

  6. 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

  7. 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

  8. 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

  9. 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)

  10. Tevatron AC dipole system

    SciTech Connect

    Miyamoto, R.; Kopp, S.E.; Jansson, A.; Syphers, M.J.; /Fermilab

    2007-06-01

    The AC dipole is an oscillating dipole magnet which can induce large amplitude oscillations without the emittance growth and decoherence. These properties make it a good tool to measure optics of a hadron synchrotron. The vertical AC dipole for the Tevatron is powered by an inexpensive high power audio amplifier since its operating frequency is approximately 20 kHz. The magnet is incorporated into a parallel resonant system to maximize the current. The use of a vertical pinger magnet which has been installed in the Tevatron made the cost relatively inexpensive. Recently, the initial system was upgraded with a more powerful amplifier and oscillation amplitudes up to 2-3{sigma} were achieved with the 980 GeV proton beam. This paper discusses details of the Tevatron AC dipole system and also shows its test results.

  11. 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

  12. 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.

  13. 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.

  14. ACS CCDs daily monitor

    NASA Astrophysics Data System (ADS)

    Sirianni, Marco

    2006-07-01

    This program consists of a set of basic tests to monitor, the read noise, thedevelopment of hot pixels and test for any source of noise in ACS CCDdetectors. The files, biases and dark will be used to create referencefiles for science calibration. This programme will be for the entire lifetime of ACS.For cycle 15 the program will cover 18 months 12.1.06->05.31.08and it has been divied into three different proposal each covering six months.The three poroposal are 11041-11042-11043.

  15. ac bidirectional motor controller

    NASA Technical Reports Server (NTRS)

    Schreiner, K.

    1988-01-01

    Test data are presented and the design of a high-efficiency motor/generator controller at NASA-Lewis for use with the Space Station power system testbed is described. The bidirectional motor driver is a 20 kHz to variable frequency three-phase ac converter that operates from the high-frequency ac bus being designed for the Space Station. A zero-voltage-switching pulse-density-modulation technique is used in the converter to shape the low-frequency output waveform.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. AC/DC converter

    NASA Astrophysics Data System (ADS)

    Jain, Praveen K.

    1992-08-01

    In a system such as a 20 kHz space station primary electrical power distribution system, power conversion from AC to DC is required. Some of the basic requirements for this conversion are high efficiency, light weight and small volume, regulated output voltage, close to unity input power factor, distortionless input current, soft-starting, low electromagnetic interference, and high reliability. An AC-to-DC converter is disclosed which satisfies the main design objectives of such converters for use in space. The converter of the invention comprises an input transformer, a resonant network, a current controller, a diode rectifier, and an output filter. The input transformer is for connection to a single phase, high frequency, sinusoidal waveform AC voltage source and provides a matching voltage isolating from the AC source. The resonant network converts this voltage to a sinusoidal, high frequency bidirectional current output, which is received by the current controller to provide the desired output current. The diode rectifier is connected in parallel with the current controller to convert the bidirectional current into a unidirectional current output. The output filter is connected to the rectifier to provide an essentially ripple-free, substantially constant voltage DC output.

  3. AC/RF Superconductivity

    SciTech Connect

    Ciovati, Gianluigi

    2015-02-01

    This contribution provides a brief introduction to AC/RF superconductivity, with an emphasis on application to accelerators. The topics covered include the surface impedance of normal conductors and superconductors, the residual resistance, the field dependence of the surface resistance, and the superheating field.

  4. Neurodegeneration in a Drosophila model of adrenoleukodystrophy: the roles of the Bubblegum and Double bubble acyl-CoA synthetases

    PubMed Central

    Sivachenko, Anna; Gordon, Hannah B.; Kimball, Suzanne S.; Gavin, Erin J.; Bonkowsky, Joshua L.; Letsou, Anthea

    2016-01-01

    ABSTRACT Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6 (encoding a very-long-chain ACS), a human homolog of bgm and dbb. PMID:26893370

  5. Reduced density of glutamine synthetase immunoreactive astrocytes in different cortical areas in major depression but not in bipolar I disorder

    PubMed Central

    Bernstein, Hans-Gert; Meyer-Lotz, Gabriela; Dobrowolny, Henrik; Bannier, Jana; Steiner, Johann; Walter, Martin; Bogerts, Bernhard

    2015-01-01

    There is increasing evidence for disturbances within the glutamate system in patients with affective disorders, which involve disruptions of the glutamate–glutamine-cycle. The mainly astroglia-located enzyme glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a central role in glutamate and glutamine homoeostasis. However, GS is also expressed in numerous oligodendrocytes (OLs), another class of glial cells implicated in mood disorder pathology. To learn more about the role of glia-associated GS in mental illnesses, we decided to find out if numerical densities of glial cells immunostained for the enzyme protein differ between subjects with major depressive disorder, bipolar disorder (BD), and psychically healthy control cases. Counting of GS expressing astrocytes (ACs) and OLs in eight cortical and two subcortical brain regions of subjects with mood disorder (N = 14), BD (N = 15), and controls (N = 16) revealed that in major depression the densities of ACs were significantly reduced in some cortical but not subcortical gray matter areas, whereas no changes were found for OLs. In BD no alterations of GS-immunoreactive glia were found. From our findings we conclude that (1) GS expressing ACs are prominently involved in glutamate-related disturbances in major depression, but not in BD and (2) GS expressing OLs, though being present in significant numbers in prefrontal cortical areas, play a minor (if any) role in mood disorder pathology. The latter assumption is supported by findings of others showing that – at least in the mouse brain cortex – GS immunoreactive oligodendroglial cells are unable to contribute to the glutamate–glutamine-cycle due to the complete lack of amino acid transporters (Takasaki et al., 2010). PMID:26321908

  6. Reduced density of glutamine synthetase immunoreactive astrocytes in different cortical areas in major depression but not in bipolar I disorder.

    PubMed

    Bernstein, Hans-Gert; Meyer-Lotz, Gabriela; Dobrowolny, Henrik; Bannier, Jana; Steiner, Johann; Walter, Martin; Bogerts, Bernhard

    2015-01-01

    There is increasing evidence for disturbances within the glutamate system in patients with affective disorders, which involve disruptions of the glutamate-glutamine-cycle. The mainly astroglia-located enzyme glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a central role in glutamate and glutamine homoeostasis. However, GS is also expressed in numerous oligodendrocytes (OLs), another class of glial cells implicated in mood disorder pathology. To learn more about the role of glia-associated GS in mental illnesses, we decided to find out if numerical densities of glial cells immunostained for the enzyme protein differ between subjects with major depressive disorder, bipolar disorder (BD), and psychically healthy control cases. Counting of GS expressing astrocytes (ACs) and OLs in eight cortical and two subcortical brain regions of subjects with mood disorder (N = 14), BD (N = 15), and controls (N = 16) revealed that in major depression the densities of ACs were significantly reduced in some cortical but not subcortical gray matter areas, whereas no changes were found for OLs. In BD no alterations of GS-immunoreactive glia were found. From our findings we conclude that (1) GS expressing ACs are prominently involved in glutamate-related disturbances in major depression, but not in BD and (2) GS expressing OLs, though being present in significant numbers in prefrontal cortical areas, play a minor (if any) role in mood disorder pathology. The latter assumption is supported by findings of others showing that - at least in the mouse brain cortex - GS immunoreactive oligodendroglial cells are unable to contribute to the glutamate-glutamine-cycle due to the complete lack of amino acid transporters (Takasaki et al., 2010).

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases.

    PubMed

    Tong, Fumin; Black, Paul N; Coleman, Rosalind A; DiRusso, Concetta C

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.

  13. 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

  14. 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.

  15. 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)

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. AC resistance measuring instrument

    DOEpatents

    Hof, P.J.

    1983-10-04

    An auto-ranging AC resistance measuring instrument for remote measurement of the resistance of an electrical device or circuit connected to the instrument includes a signal generator which generates an AC excitation signal for application to a load, including the device and the transmission line, a monitoring circuit which provides a digitally encoded signal representing the voltage across the load, and a microprocessor which operates under program control to provide an auto-ranging function by which range resistance is connected in circuit with the load to limit the load voltage to an acceptable range for the instrument, and an auto-compensating function by which compensating capacitance is connected in shunt with the range resistance to compensate for the effects of line capacitance. After the auto-ranging and auto-compensation functions are complete, the microprocessor calculates the resistance of the load from the selected range resistance, the excitation signal, and the load voltage signal, and displays of the measured resistance on a digital display of the instrument. 8 figs.

  7. AC Resistance measuring instrument

    DOEpatents

    Hof, Peter J.

    1983-01-01

    An auto-ranging AC resistance measuring instrument for remote measurement of the resistance of an electrical device or circuit connected to the instrument includes a signal generator which generates an AC excitation signal for application to a load, including the device and the transmission line, a monitoring circuit which provides a digitally encoded signal representing the voltage across the load, and a microprocessor which operates under program control to provide an auto-ranging function by which range resistance is connected in circuit with the load to limit the load voltage to an acceptable range for the instrument, and an auto-compensating function by which compensating capacitance is connected in shunt with the range resistance to compensate for the effects of line capacitance. After the auto-ranging and auto-compensation functions are complete, the microprocessor calculates the resistance of the load from the selected range resistance, the excitation signal, and the load voltage signal, and displays of the measured resistance on a digital display of the instrument.

  8. AC Optimal Power Flow

    SciTech Connect

    2016-10-04

    In this work, we have implemented and developed the simulation software to implement the mathematical model of an AC Optimal Power Flow (OPF) problem. The objective function is to minimize the total cost of generation subject to constraints of node power balance (both real and reactive) and line power flow limits (MW, MVAr, and MVA). We have currently implemented the polar coordinate version of the problem. In the present work, we have used the optimization solver, Knitro (proprietary and not included in this software) to solve the problem and we have kept option for both the native numerical derivative evaluation (working satisfactorily now) as well as for analytical formulas corresponding to the derivatives being provided to Knitro (currently, in the debugging stage). Since the AC OPF is a highly non-convex optimization problem, we have also kept the option for a multistart solution. All of these can be decided by the user during run-time in an interactive manner. The software has been developed in C++ programming language, running with GCC compiler on a Linux machine. We have tested for satisfactory results against Matpower for the IEEE 14 bus system.

  9. Identification of /sup 233/Ac

    SciTech Connect

    Chu, Y.Y.; Zhou, M.L.

    1983-09-01

    We report in this paper identification of the new isotope /sup 233/Ac. Uranium targets were irradiated with 28 GeV protons; after rapid retrieval of the target and separation of actinium from thorium, /sup 233/Ac was allowed to decay into the known /sup 233/Th daughter. Exhaustive chemical purification was employed to permit the identification of /sup 233/Th via its characteristic ..gamma.. radiations. The half-life derived for /sup 233/Ac from several experiments is 2.3 +- 0.3 min. The production cross section for /sup 233/Ac is 100 ..mu..b.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

    PubMed

    Novakovic, Predrag; Huang, Yanyun Y; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R; Middleton, Dorothy M; Loewen, Matthew E; Kidney, Beverly A; Simko, Elemir

    2015-04-01

    F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.

  15. Digital ac monitor

    DOEpatents

    Hart, George W.; Kern, Jr., Edward C.

    1987-06-09

    An apparatus and method is provided for monitoring a plurality of analog ac circuits by sampling the voltage and current waveform in each circuit at predetermined intervals, converting the analog current and voltage samples to digital format, storing the digitized current and voltage samples and using the stored digitized current and voltage samples to calculate a variety of electrical parameters; some of which are derived from the stored samples. The non-derived quantities are repeatedly calculated and stored over many separate cycles then averaged. The derived quantities are then calculated at the end of an averaging period. This produces a more accurate reading, especially when averaging over a period in which the power varies over a wide dynamic range. Frequency is measured by timing three cycles of the voltage waveform using the upward zero crossover point as a starting point for a digital timer.

  16. Digital ac monitor

    DOEpatents

    Hart, G.W.; Kern, E.C. Jr.

    1987-06-09

    An apparatus and method is provided for monitoring a plurality of analog ac circuits by sampling the voltage and current waveform in each circuit at predetermined intervals, converting the analog current and voltage samples to digital format, storing the digitized current and voltage samples and using the stored digitized current and voltage samples to calculate a variety of electrical parameters; some of which are derived from the stored samples. The non-derived quantities are repeatedly calculated and stored over many separate cycles then averaged. The derived quantities are then calculated at the end of an averaging period. This produces a more accurate reading, especially when averaging over a period in which the power varies over a wide dynamic range. Frequency is measured by timing three cycles of the voltage waveform using the upward zero crossover point as a starting point for a digital timer. 24 figs.

  17. Cooling Floor AC Systems

    NASA Astrophysics Data System (ADS)

    Jun, Lu; Hao, Ding; Hong, Zhang; Ce, Gao Dian

    The present HVAC equipments for the residential buildings in the Hot-summer-and-Cold-winter climate region are still at a high energy consuming level. So that the high efficiency HVAC system is an urgently need for achieving the preset government energy saving goal. With its advantage of highly sanitary, highly comfortable and uniform of temperature field, the hot-water resource floor radiation heating system has been widely accepted. This paper has put forward a new way in air-conditioning, which combines the fresh-air supply unit and such floor radiation system for the dehumidification and cooling in summer or heating in winter. By analyze its advantages and limitations, we found that this so called Cooling/ Heating Floor AC System can improve the IAQ of residential building while keep high efficiency quality. We also recommend a methodology for the HVAC system designing, which will ensure the reduction of energy cost of users.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  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. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Automated ac galvanomagnetic measurement system

    NASA Technical Reports Server (NTRS)

    Szofran, F. R.; Espy, P. N.

    1985-01-01

    An automated, ac galvanomagnetic measurement system is described. Hall or van der Pauw measurements in the temperature range 10-300 K can be made at a preselected magnetic field without operator attendance. Procedures to validate sample installation and correct operation of other system functions, such as magnetic field and thermometry, are included. Advantages of ac measurements are discussed.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. [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)

  15. 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

  16. 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

  17. 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

  18. Ontogeny of mRNA expression and activity of long-chain acyl-CoA synthetase (ACSL) isoforms in Mus musculus heart.

    PubMed

    de Jong, Hendrik; Neal, Andrea C; Coleman, Rosalind A; Lewin, Tal M

    2007-01-01

    Long-chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for virtually every metabolic pathway that catabolizes FA or synthesizes complex lipids. We have hypothesized that each of the five cloned ACSL isoforms partitions FA towards specific downstream pathways. Adult heart expresses all five cloned ACSL isoforms, but their independent functional roles have not been elucidated. Studies implicate ACSL1 in both oxidative and lipid synthetic pathways. To clarify the functional role of ACSL1 and the other ACSL isoforms (3-6), we examined ACS specific activity and Acsl mRNA expression in the developing mouse heart which increases FA oxidative pathways for energy production after birth. Compared to the embryonic heart, ACS specific activity was 14-fold higher on post-natal day 1 (P1). On P1, as compared to the fetus, only Acsl1 mRNA increased, whereas transcripts for the other Acsl isoforms remained the same, suggesting that ACSL1 is the major isoform responsible for activating long-chain FA for myocardial oxidation after birth. In contrast, the mRNA abundance of Acsl3 was highest on E16, and decreased dramatically by P7, suggesting that ACSL3 may play a critical role during the development of the fetal heart. Our data support the hypothesis that each ACSL has a specific role in the channeling of FA towards distinct metabolic fates.

  19. Involvement of acyl-CoA synthetase genes in n-alkane assimilation and fatty acid utilization in yeast Yarrowia lipolytica.

    PubMed

    Tenagy; Park, Jun Seok; Iwama, Ryo; Kobayashi, Satoshi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-06-01

    Here, we investigated the roles of YAL1 (FAA1) and FAT1 encoding acyl-CoA synthetases (ACSs) and three additional orthologs of ACS genes FAT2-FAT4 of the yeast Yarrowia lipolytica in the assimilation or utilization of n-alkanes and fatty acids. ACS deletion mutants were generated to characterize their function. The FAT1 deletion mutant exhibited decreased growth on n-alkanes of 10-18 carbons, whereas the FAA1 mutant showed growth reduction on n-alkane of 16 carbons. However, FAT2-FAT4 deletion mutants did not show any growth defects, suggesting that FAT1 and FAA1 are involved in the activation of fatty acids produced during the metabolism of n-alkanes. In contrast, deletions of FAA1 and FAT1-FAT4 conferred no defect in growth on fatty acids. The wild-type strain grew in the presence of cerulenin, an inhibitor of fatty acid synthesis, by utilizing exogenously added fatty acid or fatty acid derived from n-alkane when oleic acid or n-alkane of 18 carbons was supplemented. However, the FAA1 deletion mutant did not grow, indicating a critical role for FAA1 in the utilization of fatty acids. Fluorescent microscopic observation and biochemical analyses suggested that Fat1p is present in the peroxisome and Faa1p is localized in the cytosol and to membranes.

  20. Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis.

    PubMed

    Maloberti, Paula; Castilla, Rocío; Castillo, Fernanda; Cornejo Maciel, Fabiana; Mendez, Carlos F; Paz, Cristina; Podestá, Ernesto J

    2005-04-01

    Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

  1. Layoff Handling Still Lags ACS Standards.

    ERIC Educational Resources Information Center

    Chemical and Engineering News, 1981

    1981-01-01

    Reviews termination procedures of professional chemists and the compliance of these terminations to the American Chemical Society's (ACS's) Professional Employment Guidelines. Provides the ACS guidelines. (DS)

  2. AC photovoltaic module magnetic fields

    SciTech Connect

    Jennings, C.; Chang, G.J.; Reyes, A.B.; Whitaker, C.M.

    1997-12-31

    Implementation of alternating current (AC) photovoltaic (PV) modules, particularly for distributed applications such as PV rooftops and facades, may be slowed by public concern about electric and magnetic fields (EMF). This paper documents magnetic field measurements on an AC PV module, complementing EMF research on direct-current PV modules conducted by PG and E in 1993. Although not comprehensive, the PV EMF data indicate that 60 Hz magnetic fields (the EMF type of greatest public concern) from PV modules are comparable to, or significantly less than, those from household appliances. Given the present EMF research knowledge, AC PV module EMF may not merit considerable concern.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  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. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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...

  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. 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.

  6. 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

  7. NONLINEAR DIAGNOSTICS USING AC DIPOLES.

    SciTech Connect

    PEGGS,S.

    1999-03-29

    There are three goals in the accurate nonlinear diagnosis of a storage ring. First, the beam must be moved to amplitudes many times the natural beam size. Second, strong and long lasting signals must be generated. Third, the measurement technique should be non-destructive. Conventionally, a single turn kick moves the beam to large amplitudes, and turn-by-turn data are recorded from multiple beam position monitors (BPMs) [1-6]. Unfortunately, tune spread across the beam causes the center of charge beam signal to ''decohere'' on a time scale often less than 100 turns. Filamentation also permanently destroys the beam emittance (in a hadron ring). Thus, the ''strong single turn kick'' technique successfully achieves only one out of the three goals. AC dipole techniques can achieve all three. Adiabatically excited AC dipoles slowly move the beam out to large amplitudes. The coherent signals then recorded last arbitrarily long. The beam maintains its original emittance if the AC dipoles are also turned off adiabatically, ready for further use. The AGS already uses an RF dipole to accelerate polarized proton beams through depolarizing resonances with minimal polarization loss [7]. Similar AC dipoles will be installed in the horizontal and vertical planes of both rings in RHIC [8]. The RHIC AC dipoles will also be used as spin flippers, and to measure linear optical functions [9].

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. ACS from development to operations

    NASA Astrophysics Data System (ADS)

    Caproni, Alessandro; Colomer, Pau; Jeram, Bogdan; Sommer, Heiko; Chiozzi, Gianluca; Mañas, Miguel M.

    2016-08-01

    The ALMA Common Software (ACS), provides the infrastructure of the distributed software system of ALMA and other projects. ACS, built on top of CORBA and Data Distribution Service (DDS) middleware, is based on a Component- Container paradigm and hides the complexity of the middleware allowing the developer to focus on domain specific issues. The transition of the ALMA observatory from construction to operations brings with it that ACS effort focuses primarily on scalability, stability and robustness rather than on new features. The transition came together with a shorter release cycle and a more extensive testing. For scalability, the most problematic area has been the CORBA notification service, used to implement the publisher subscriber pattern because of the asynchronous nature of the paradigm: a lot of effort has been spent to improve its stability and recovery from run time errors. The original bulk data mechanism, implemented using the CORBA Audio/Video Streaming Service, showed its limitations and has been replaced with a more performant and scalable DDS implementation. Operational needs showed soon the difference between releases cycles for Online software (i.e. used during observations) and Offline software, which requires much more frequent releases. This paper attempts to describe the impact the transition from construction to operations had on ACS, the solution adopted so far and a look into future evolution.

  14. Simple Equipment for Imaging AC.

    ERIC Educational Resources Information Center

    Kamata, Masahiro; Anayama, Takayuki

    2003-01-01

    Presents an effective way to demonstrate the difference between direct current and alternating current using red and green LEDs. Describes how to make a tool that shows how an AC voltage changes with time using the afterimage effect of the LEDs. (Author/NB)

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  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. 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

  14. 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.

  15. 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.

  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. 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

  18. 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.

  19. ac-resistance-measuring instrument

    SciTech Connect

    Hof, P.J.

    1981-04-22

    An auto-ranging ac resistance measuring instrument for remote measurement of the resistance of an electrical device or circuit connected to the instrument includes a signal generator which generates an ac excitation signal for application to a load, including the device and the transmission line, a monitoring circuit which provides a digitally encoded signal representing the voltage across the load, and a microprocessor which operates under program control to provide an auto-ranging function by which range resistance is connected in circuit with the load to limit the load voltage to an acceptable range for the instrument, and an auto-compensating function by which compensating capacitance is connected in shunt with the range resistance to compensate for the effects of line capacitance.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.,

  14. 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

  15. 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

  16. Defects in the C. elegans acyl-CoA synthase, acs-3, and nuclear hormone receptor, nhr-25, cause sensitivity to distinct, but overlapping stresses.

    PubMed

    Ward, Jordan D; Mullaney, Brendan; Schiller, Benjamin J; He, Le D; Petnic, Sarah E; Couillault, Carole; Pujol, Nathalie; Bernal, Teresita U; Van Gilst, Marc R; Ashrafi, Kaveh; Ewbank, Jonathan J; Yamamoto, Keith R

    2014-01-01

    Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development.

  17. Simultaneous distribution of AC and DC power

    DOEpatents

    Polese, Luigi Gentile

    2015-09-15

    A system and method for the transport and distribution of both AC (alternating current) power and DC (direct current) power over wiring infrastructure normally used for distributing AC power only, for example, residential and/or commercial buildings' electrical wires is disclosed and taught. The system and method permits the combining of AC and DC power sources and the simultaneous distribution of the resulting power over the same wiring. At the utilization site a complementary device permits the separation of the DC power from the AC power and their reconstruction, for use in conventional AC-only and DC-only devices.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis.

    PubMed

    Takasaki, Kazuto; Shoun, Hirofumi; Yamaguchi, Masashi; Takeo, Kanji; Nakamura, Akira; Hoshino, Takayuki; Takaya, Naoki

    2004-03-26

    Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.

  3. 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.

  4. Effect of the magnetic material on AC losses in HTS conductors in AC magnetic field carrying AC transport current

    NASA Astrophysics Data System (ADS)

    Wan, Xing-Xing; Huang, Chen-Guang; Yong, Hua-Dong; Zhou, You-He

    2015-11-01

    This paper presents an investigation on the AC losses in several typical superconducting composite conductors using the H-formulation model. A single superconducting strip with ferromagnetic substrate or cores and a stack of coated conductors with ferromagnetic substrates are studied. We consider all the coated conductors carrying AC transport currents and simultaneously exposed to perpendicular AC magnetic fields. The influences of the amplitude, frequency, phase difference and ferromagnetic materials on the AC losses are investigated. The results show that the magnetization losses of single strip and stacked strips have similar characteristics. The ferromagnetic substrate can increase the magnetization loss at low magnetic field, and decrease the loss at high magnetic field. The ferromagnetic substrate can obviously increase the transport loss in stacked strips. The trends of total AC losses of single strip and stacked strips are similar when they are carrying current or exposed to a perpendicular magnetic field. The effect of the frequency on the total AC losses of single strip is related to the amplitude of magnetic field. The AC losses decrease with increasing frequency in low magnetic field region while increase in high magnetic field region. As the phase difference changes, there is a periodic variation for the AC losses. Moreover, when the strip is under only the transport current and magnetic field, the ferromagnetic cores will increase the AC losses for large transport current or field.

  5. 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

  6. 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.

  7. ac electroosmosis in rectangular microchannels.

    PubMed

    Campisi, Michele; Accoto, Dino; Dario, Paolo

    2005-11-22

    Motivated by the growing interest in ac electroosmosis as a reliable no moving parts strategy to control fluid motion in microfluidic devices for biomedical applications, such as lab-on-a-chip, we study transient and steady-state electrokinetic phenomena (electroosmosis and streaming currents) in infinitely extended rectangular charged microchannels. With the aid of Fourier series and Laplace transforms we provide a general formal solution of the problem, which is used to study the time-dependent response to sudden ac applied voltage differences in case of finite electric double layer. The Debye-Huckel approximation has been adopted to allow for an algebraic solution of the Poisson-Boltzmann problem in Fourier space. We obtain the expressions of flow velocity profiles, flow rates, streaming currents, as well as expressions of the complex hydraulic and electrokinetic conductances. We analyze in detail the dependence of the electrokinetic conductance on the extension of linear dimensions relative to the Debye length, with an eye on finite electric double layer effects.

  8. ACS PSF Variations with Temperatures

    NASA Astrophysics Data System (ADS)

    Sahu, Kailash C.; Lallo, Matt; Makidon, Russ

    2007-09-01

    We have used the HST ACS/WFC observations of a Galactic bulge field taken over a continuous interval of 7 days (Prop 9750) to investigate the possible dependence of the ACS focus with the external temperatures. This dataset allows us to investigate possible focus variations over timescales of a few hours to a few days. The engineering data related to the external temperatures for this duration show that the maximum temperature change occurred over the first 1.5 days. Among all the different temperatures recorded, the truss diametric differential and the truss axial temperatures are the only two temperatures which have the same timescale of variation as the PSFwidth variations. The PSF-widths also strongly correlate with these two temperatures during this time interval. We empirically fit the PSF-width variations with these 2 temperature sensor values. This suggests that the focus has a similar dependence, and we recommend that this finding be followed up with the determination of actual focus values to check if the focus values indeed have the same correlation. If so, the temperature data can be useful in estimating the focus values, which can then be used to predict the PSFs to a first order.

  9. ac electroosmosis in rectangular microchannels

    NASA Astrophysics Data System (ADS)

    Campisi, Michele; Accoto, Dino; Dario, Paolo

    2005-11-01

    Motivated by the growing interest in ac electroosmosis as a reliable no moving parts strategy to control fluid motion in microfluidic devices for biomedical applications, such as lab-on-a-chip, we study transient and steady-state electrokinetic phenomena (electroosmosis and streaming currents) in infinitely extended rectangular charged microchannels. With the aid of Fourier series and Laplace transforms we provide a general formal solution of the problem, which is used to study the time-dependent response to sudden ac applied voltage differences in case of finite electric double layer. The Debye-Hückel approximation has been adopted to allow for an algebraic solution of the Poisson-Boltzmann problem in Fourier space. We obtain the expressions of flow velocity profiles, flow rates, streaming currents, as well as expressions of the complex hydraulic and electrokinetic conductances. We analyze in detail the dependence of the electrokinetic conductance on the extension of linear dimensions relative to the Debye length, with an eye on finite electric double layer effects.

  10. RHIC spin flipper AC dipole controller

    SciTech Connect

    Oddo, P.; Bai, M.; Dawson, C.; Gassner, D.; Harvey, M.; Hayes, T.; Mernick, K.; Minty, M.; Roser, T.; Severino, F.; Smith, K.

    2011-03-28

    The RHIC Spin Flipper's five high-Q AC dipoles which are driven by a swept frequency waveform require precise control of phase and amplitude during the sweep. This control is achieved using FPGA based feedback controllers. Multiple feedback loops are used to and dynamically tune the magnets. The current implementation and results will be presented. Work on a new spin flipper for RHIC (Relativistic Heavy Ion Collider) incorporating multiple dynamically tuned high-Q AC-dipoles has been developed for RHIC spin-physics experiments. A spin flipper is needed to cancel systematic errors by reversing the spin direction of the two colliding beams multiple times during a store. The spin flipper system consists of four DC-dipole magnets (spin rotators) and five AC-dipole magnets. Multiple AC-dipoles are needed to localize the driven coherent betatron oscillation inside the spin flipper. Operationally the AC-dipoles form two swept frequency bumps that minimize the effect of the AC-dipole dipoles outside of the spin flipper. Both AC bumps operate at the same frequency, but are phase shifted from each other. The AC-dipoles therefore require precise control over amplitude and phase making the implementation of the AC-dipole controller the central challenge.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  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. 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.

  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. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.

    PubMed

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; van Voorhis, Wesley C; Hol, Wim G J

    2010-03-26

    Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Three phase AC motor controller

    DOEpatents

    Vuckovich, Michael; Wright, Maynard K.; Burkett, John P.

    1984-03-20

    A motor controller for a three phase AC motor (10) which is adapted to operate bidirectionally from signals received either from a computer (30) or a manual control (32). The controller is comprised of digital logic circuit means which implement a forward and reverse command signal channel (27, 29) for the application of power through the forward and reverse power switching relays (16, 18, 20, 22). The digital logic elements are cross coupled to prevent activation of both channels simultaneously and each includes a plugging circuit (65, 67) for stopping the motor upon the removal of control signal applied to one of the two channels (27, 29) for a direction of rotation desired. Each plugging circuit (65, 67) includes a one-shot pulse signal generator (88, 102) which outputs a single pulse signal of predetermined pulsewidth which is adapted to inhibit further operation of the application of power in the channel which is being activated and to apply a reversal command signal to the other channel which provides a reversed phase application of power to the motor for a period defined by the pulse-width output of the one-shot signal generator to plug the motor (10) which will then be inoperative until another rotational command signal is applied to either of the two channels.

  10. Memory effect in ac plasma displays

    NASA Astrophysics Data System (ADS)

    Szlenk, K.; Obuchowicz, E.

    1993-10-01

    The bistable or `memory' mode of operation of an ac plasma display panel is presented. The difference between dc and ac plasma panel operation from the point of view of memory function is discussed. The graphic ac plasma display with thin film Cr-Cu-Cr electrodes was developed in OBREP and its basic parameters are described. It consists of 36 X 59 picture elements, its outer dimensions are: 76 X 52 mm2 and the screen size is: 49 X 30 mm2. The different dielectric glass materials were applied as dielectric layers and the influence of the properties of these materials on display parameters and memory function was investigated.

  11. Superconductor coil geometry and ac losses

    NASA Technical Reports Server (NTRS)

    Pierce, T. V., Jr.; Zapata, R. N.

    1976-01-01

    An empirical relation is presented which allows simple computation of volume-averaged winding fields from central fields for coils of small rectangular cross sections. This relation suggests that, in certain applications, ac-loss minimization can be accomplished by use of low winding densities, provided that hysteresis losses are independent of winding density. The ac-loss measurements on coils wound of twisted multifilamentary composite superconductors show no significant dependence on ac losses on winding density, thus permitting the use of winding density as an independent design parameter in loss minimization.

  12. Exenatide: AC 2993, AC002993, AC2993A, exendin 4, LY2148568.

    PubMed

    2004-01-01

    Exenatide [AC002993, AC2993A, AC 2993, LY2148568, exendin 4], a glucagon-like peptide-1 (GLP-1) agonist, is a synthetic exendin 4 compound under development with Amylin Pharmaceuticals for the treatment of type 2 diabetes. Both exendin 4 and its analogue, exendin 3, are 39-amino acid peptides isolated from Heloderma horridum lizard venom that have different amino acids at positions 2 and 3, respectively. Exendins are able to stimulate insulin secretion in response to rising blood glucose levels, and modulate gastric emptying to slow the entry of ingested sugars into the bloodstream. Amylin Pharmaceuticals acquired exclusive patent rights for the two exendin compounds (exendin 3 and exendin 4) from the originator, Dr John Eng (Bronx, NY, US). On 20 September 2002, Amylin and Eli Lilly signed a collaborative agreement for the development and commercialisation of exenatide for type 2 diabetes. Under the terms of the agreement, Eli Lilly has paid Amylin a licensing fee of 80 million US dollars and bought Amylin's stock worth 30 million US dollars at 18.69 US dollars a share. After the initial payment, Eli Lilly will pay Amylin up to 85 US dollars million upon reaching certain milestones and also make an additional payment of up to 130 million US dollars upon global commercialisation of exenatide. Both companies will share the US development and commercialisation costs, while Eli Lilly will pick up up to 80% of development costs and all commercialisation costs outside the US. Amylin and Eli Lilly will equally share profit from sales in the US, while Eli Lilly will get 80% of the profit outside the US and Amylin will get the rest. This agreement has also enabled Amylin to train its sales force to co-promote Lilly's human growth hormone Humatrope. Alkermes will receive research and development funding and milestone payments, and also a combination of royalty payments and manufacturing fees based on product sales. Alkermes undertakes the responsibility for the development

  13. 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

  14. 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.

  15. 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.

  16. High School Teachers Win ACS Prizes

    NASA Astrophysics Data System (ADS)

    Editorial Staff, Jce

    2009-07-01

    William E. Snyder is the 2009 winner of the ACS Division of Chemical Education Central Region Award for Excellence in High School Teaching; Sally Mitchell is the winner of the 2009 James Bryant Conant Award in High School Chemistry Teaching.

  17. The AC-120: The advanced commercial transport

    NASA Technical Reports Server (NTRS)

    Duran, David; Griffin, Ernest; Mendoza, Saul; Nguyen, Son; Pickett, Tim; Noernberg, Clemm

    1993-01-01

    The main objective of this design was to fulfill a need for a new airplane to replace the aging 100 to 150 passenger, 1500 nautical mile range aircraft such as the Douglas DC9 and Boeing 737-100 airplanes. After researching the future aircraft market, conducting extensive trade studies, and analysis on different configurations, the AC-120 Advanced Commercial Transport final design was achieved. The AC-120's main design features include the incorporation of a three lifting surface configuration which is powered by two turboprop engines. The AC-120 is an economically sensitive aircraft which meets the new FM Stage Three noise requirements, and has lower NO(x) emissions than current turbofan powered airplanes. The AC-120 also improves on its contemporaries in passenger comfort, manufacturing, and operating cost.

  18. Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases.

    PubMed

    Bloudoff, Kristjan; Alonzo, Diego A; Schmeing, T Martin

    2016-03-17

    Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

  19. p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates.

    PubMed

    Nguyen, Thang Van; Li, Jing; Lu, Chin-Chun Jean; Mamrosh, Jennifer L; Lu, Gang; Cathers, Brian E; Deshaies, Raymond J

    2017-04-04

    Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4(CRBN) and p97 pathways.

  20. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    PubMed Central

    Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A.

    2009-01-01

    FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokary­otic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively. PMID:20054130

  1. Plant growth is influenced by glutamine synthetase-catalyzed nitrogen metabolism

    SciTech Connect

    Langston-Unkefer, P.J.

    1991-06-11

    Ammonia assimilation has been implicated as participating in regulation of nitrogen fixation in free-living bacteria. In fact, these simple organisms utilize an integrated regulation of carbon and nitrogen metabolism; we except to observe an integration of nitrogen and carbon fixation in plants; how could these complex systems grow efficiently and compete in the ecosystem without coordinating these two crucial activities We have been investigating the role of ammonia assimilation regulating the complex symbiotic nitrogen fixation of legumes. Just as is observed in the simple bacterial systems, perturbation of ammonia assimilation in legumes results in increased overall nitrogen fixation. The perturbed plants have increased growth and total nitrogen fixation capability. Because we have targeted the first enyzme in ammonia assimilation, glutamine synthetase, this provides a marker that could be used to assist selection or screening for increased biomass yield. 45 refs., 4 tabs.

  2. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    PubMed Central

    Pimentel-Elardo, Sheila Marie; Grozdanov, Lubomir; Proksch, Sebastian; Hentschel, Ute

    2012-01-01

    Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis. PMID:22822366

  3. 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.

  4. Heterogenous expression of poly-gamma-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3.

    PubMed

    Wang, N; Yang, G; Che, C; Liu, Y

    2011-01-01

    Bacillus licheniformis WBL-3, one of poly-gamma-glutamic acid (gamma-PGA) producers, depends on the existence of glutamate in the medium. In this paper, gamma-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgsBCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IF03336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced gamma-PGA extracellularly. The yield of gamma-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar gamma-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize gamma-PGA.

  5. Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions.

    PubMed

    Harden, Bradley J; Frueh, Dominique P

    2017-01-24

    Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.

  6. Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

    PubMed Central

    Betti, Marco; García-Calderón, Margarita; Pérez-Delgado, Carmen M.; Credali, Alfredo; Estivill, Guillermo; Galván, Francisco; Vega, José M.; Márquez, Antonio J.

    2012-01-01

    Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism. PMID:22942686

  7. S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth1[OPEN

    PubMed Central

    Zou, Ting

    2016-01-01

    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. Here, we show that mat3 mutants have impaired pollen tube growth and reduced seed set. Metabolomics analyses confirmed that mat3 pollen and pollen tubes overaccumulate Met and that mat3 pollen has several metabolite profiles, such as those of polyamine biosynthesis, which are different from those of the wild type. Additionally, we show that disruption of Met metabolism in mat3 pollen affected transfer RNA and histone methylation levels. Thus, our results suggest a connection between metabolism and epigenetics. PMID:27482079

  8. Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation

    PubMed Central

    Ballut, Lionel; Violot, Sébastien; Shivakumaraswamy, Santosh; Thota, Lakshmi Prasoona; Sathya, Manu; Kunala, Jyothirmai; Dijkstra, Bauke W.; Terreux, Raphaël; Haser, Richard; Balaram, Hemalatha; Aghajari, Nushin

    2015-01-01

    GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371–375 holding catalytic residues and in loop 376–401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme. PMID:26592566

  9. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  10. Barley chloroplast glutamine synthetase activity is not affected by CO sub 2 -concentration

    SciTech Connect

    Avila, C.; Forde, B.; Wallsgrove, R. )

    1990-05-01

    It has been reported that when photorespiration is suppressed by raising the concentration of CO{sub 2}, the expression of the chloroplast glutamine synthetase (GS2) gene in pea leaves is reduced (Plant Cell, 1, 241). We have examined this effect in barley (Hordeum vulgare), and confirm that plants grown continuously in 0.8% CO{sub 2}, or transferred to such conditions after growth in air, appear to have a reduced GS2 mRNA abundance. However, we were unable to detect any significant difference in the extractable GS2 activity, or any change in amount of GS2 protein (judged by Western blots). Whatever controls are operating on gS2 mRNA expression in response to changes is external CO{sub 2}, they do not affect the activity or amount of the enzyme in barley.

  11. CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis

    PubMed Central

    Pan, Weijun; Pham, Van N.; Stratman, Amber N.; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R.; Lo, Brigid D.; Shaw, Kenna M.; Torres-Vazquez, Jesus; Mikelis, Constantinos M.; Gutkind, J. Silvio; Davis, George E.

    2012-01-01

    Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis. PMID:22649102

  12. ACR11 is an Activator of Plastid-Type Glutamine Synthetase GS2 in Arabidopsis thaliana.

    PubMed

    Osanai, Takashi; Kuwahara, Ayuko; Otsuki, Hitomi; Saito, Kazuki; Yokota Hirai, Masami

    2017-03-06

    Glutamine synthetase (GS) is an important enzyme for nitrogen assimilation, and GS2, encoded by GLN2, is the only plastid-type GS in Arabidopsis thaliana. A co-expression analysis suggested that the expression level of the gene encoding a uridylyltransferase-like protein, ACR11, is strongly correlated with GLN2 expression levels. Here we showed that the recombinant ACR11 protein increased GS2 activity in vitro by reducing the Km values of its substrate glutamine. A T-DNA insertion mutant of ACR11 exhibited a reduced GS activity under low nitrate conditions and reduced glutamine levels. Biochemical analyses revealed that ACR11 and GS2 interacted both in vitro and in vivo. These data demonstrate that ACR11 is an activator of GS2, giving it a mechanistic role in the nitrogen assimilation of A. thaliana.

  13. The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment.

    PubMed

    Saint-Léger, Adélaïde; Sinadinos, Christopher; Ribas de Pouplana, Lluís

    2016-04-02

    Malaria remains a major global health problem. Parasite resistance to existing drugs makes development of new antimalarials an urgency. The protein synthesis machinery is an excellent target for the development of new anti-infectives, and aminoacyl-tRNA synthetases (aaRS) have been validated as antimalarial drug targets. However, avoiding the emergence of drug resistance and improving selectivity to target aaRS in apicomplexan parasites, such as Plasmodium falciparum, remain crucial challenges. Here we discuss such issues using examples of known inhibitors of P. falciparum aaRS, namely halofuginone, cladosporin and borrelidin (inhibitors of ProRS, LysRS and ThrRS, respectively). Encouraging recent results provide useful guidelines to facilitate the development of novel drug candidates which are more potent and selective against these essential enzymes.

  14. Fluorine-19 nuclear magnetic resonance and biochemical characterization of fluorotyrosine-labeled-thymidylate-synthetase

    NASA Astrophysics Data System (ADS)

    Rosson, Dan; Lewis, Charles A.; Ellis, Paul D.; Dunlap, R. Bruce

    1994-03-01

    Fluorotyrosine has been incorporated into thymidylate synthetase from Lactobacillus casei by growth of the bacterium in media containing 3-fluorotyrosine. The enzyme exhibited a specific activity 70% of that of the normal enzyme and formed a covalent binary complex with pyrimidine nucleotides, as well as a covalent ternary complex with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate. 19F nuclear magnetic resonance spectroscopy has been used to follow the formation of these complexes. 5-Fluorodeoxyuridylate, dUMP, dTMP and dCMP produced identical conformational changes in the enzyme as monitored by the fluorotyrosyl resonances. Ternary complex formation of the fluorotyrosine-containing enzyme with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate resulted in further spectral changes.

  15. Mutational analysis of glycyl-tRNA synthetase (GARS) gene in Hirayama Disease

    PubMed Central

    Blumen, Sergiu C.; Drory, Vivian E.; Sadeh, Menachem; El-Ad, Baruch; Soimu, Uri; Groozman, Galina B.; Bouchard, Jean-Pierre; Goldfarb, Lev G.

    2009-01-01

    Sporadic juvenile muscular atrophy of the distal upper extremity or Hirayama's Disease (HD) and autosomal dominant motor distal neuronopathy/axonopathy (CMT2D/dSMA-V), produced by glycyl-tRNA synthetase (GARS) gene mutations, share some clinical features including: young age of onset, predilection for the distal upper extremity, asymmetry, sparing of proximal muscles and unusual cold sensitivity. However, incomplete penetrance of GARS gene mutations may account for apparently non-familial cases. In order to inquire whether GARS gene mutations are associated with HD we studied seven patients fulfilling the clinical and electrodiagnostic criteria for HD. All patients underwent MRI of cervical spine that excluded compressive myelopathy in neutral position and intramedullary pathology. Each patient was tested for the presence of mutations in GARS by sequencing all coding exons amplified from genomic DNA. No pathogenic mutations were found, excluding the role of GARS gene as a possible factor in the etiology of HD in this cohort. PMID:19412816

  16. The albonoursin gene Cluster of S noursei biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases.

    PubMed

    Lautru, Sylvie; Gondry, Muriel; Genet, Roger; Pernodet, Jean Luc

    2002-12-01

    Albonoursin [cyclo(deltaPhe-DeltaLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of alpha,beta unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives.

  17. An iterative nonribosomal peptide synthetase assembles the pyrrole-amide antibiotic congocidine in Streptomyces ambofaciens.

    PubMed

    Juguet, Maud; Lautru, Sylvie; Francou, François-Xavier; Nezbedová, Sárka; Leblond, Pierre; Gondry, Muriel; Pernodet, Jean-Luc

    2009-04-24

    Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.

  18. Cytosolic glutamine synthetase: a target for improvement of crop nitrogen use efficiency?

    PubMed

    Thomsen, Hanne C; Eriksson, Dennis; Møller, Inge S; Schjoerring, Jan K

    2014-10-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude that GS1 activity may be downregulated via a chain of processes elicited by metabolic imbalances and environmental constraints. We suggest that a pivotal role of GS1 may be related to the maintenance of essential nitrogen (N) flows and internal N sensing during critical stages of plant development. A number of more refined overexpression strategies exploiting gene stacking combined with tissue and cell specific targeting to overcome metabolic bottlenecks are considered along with their potential in relation to new N management strategies.

  19. Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels.

    PubMed

    Palmieri, Erika Mariana; Spera, Iolanda; Menga, Alessio; Infantino, Vittoria; Iacobazzi, Vito; Castegna, Alessandra

    2014-12-20

    The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.

  20. Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase

    NASA Technical Reports Server (NTRS)

    Martinis, S. A.; Fox, G. E.

    1997-01-01

    Recombinant E. coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine. [32P]-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging. Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent. The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively. Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism.

  1. Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics

    PubMed Central

    Chen, Yunqiu; McClure, Ryan A.; Kelleher, Neil L.

    2016-01-01

    Liquid chromatography–mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products. PMID:26831706

  2. Three-phase-to-two-phase direct AC-AC converter with three leg structure

    NASA Astrophysics Data System (ADS)

    Kwak, S.-S.

    2014-05-01

    A three-phase-to-two-phase ac-ac converter is, along with a modulation strategy based on the space vector scheme, introduced to directly drive two-phase output ac systems with high input power quality. The converter is capable of synthesising two sinusoidal output voltages with variable output frequency and arbitrary magnitude in quadrature phase-shift as well as sinusoidal input currents.

  3. Phase protection system for ac power lines

    NASA Technical Reports Server (NTRS)

    Wong, W. J. (Inventor)

    1974-01-01

    The system described provides protection for phase sensitive loads from being or remaining connected to ac power lines whenever a phase reversal occurs. It comprises a solid state phase detection circuit, a dc power relay circuit, an ac-to-dc converter for energizing the relay circuit, and a bistable four terminal transducer coupled between the phase detection circuit and the power relay circuit, for controlling both circuits.

  4. Proximal Tubule Glutamine Synthetase Expression is Necessary for the Normal Response to Dietary Protein Restriction.

    PubMed

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Verlander, Jill W; Weiner, I David

    2017-03-22

    Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and ¬decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of NH4+ and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The current study's purpose was to determine proximal tubule GS's role in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule glutamine synthetase expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

  5. Immunotherapeutic potential of N-formylated peptides of ESAT-6 and glutamine synthetase in experimental tuberculosis.

    PubMed

    Mir, Shabir Ahmad; Sharma, Sadhna

    2014-02-01

    Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host anti mycobacterial innate and/or adaptive immune response have been supposed to be useful in the treatment of tuberculosis. Since N-formyl peptides are products of bacterial metabolism, and their binding to a specific phagocyte receptor (FPR) induces chemotaxis and activation of phagocytes that are critical effectors in our innate immune system, it is reasonable to assume that the interaction between these two counterparts (i.e. formylated peptides and FPR) is also important in host defence against M. tuberculosis. In the present study the direct immunotherapeutic potential of N-formylated peptides of two non-classically secreted proteins (early secreted antigenic target-6 and glutamine synthetase) of M. tuberculosis H37Rv was evaluated. Treatment of M. tuberculosis H37Rv infected mice with N-formylated peptides of early secreted antigenic target-6 (ESAT-6) and glutamine synthetase (GS) markedly reduced the bacilli load in their lungs (p < 0.001) and spleen (p < 0.01) as compared to the untreated mice. In addition, the histopathological changes were observed to be in correlation with the CFU data with minor areas of consolidation in the lung sections of N-formylated peptide treated infected mice as compared to those of the untreated mice. Further, these N-formylated peptides were able to confer an additional therapeutic effect when given in combination with the anti tuberculosis drugs and hence can be used as an adjunct to the conventional chemotherapy against tuberculosis.

  6. Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

    PubMed Central

    Ray, P H

    1980-01-01

    3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate. PMID:6988389

  7. Ancient origin of the divergent forms of leucyl-tRNA synthetases in the Halobacteriales

    PubMed Central

    2012-01-01

    Background Horizontal gene transfer (HGT) has greatly impacted the genealogical history of many lineages, particularly for prokaryotes, with genes frequently moving in and out of a line of descent. Many genes that were acquired by a lineage in the past likely originated from ancestral relatives that have since gone extinct. During the course of evolution, HGT has played an essential role in the origin and dissemination of genetic and metabolic novelty. Results Three divergent forms of leucyl-tRNA synthetase (LeuRS) exist in the archaeal order Halobacteriales, commonly known as haloarchaea. Few haloarchaeal genomes have the typical archaeal form of this enzyme and phylogenetic analysis indicates it clusters within the Euryarchaeota as expected. The majority of sequenced halobacterial genomes possess a bacterial form of LeuRS. Phylogenetic reconstruction puts this larger group of haloarchaea at the base of the bacterial domain. The most parsimonious explanation is that an ancient transfer of LeuRS took place from an organism related to the ancestor of the bacterial domain to the haloarchaea. The bacterial form of LeuRS further underwent gene duplications and/or gene transfers within the haloarchaea, with some genomes possessing two distinct types of bacterial LeuRS. The cognate tRNALeu also reveals two distinct clusters for the haloarchaea; however, these tRNALeu clusters do not coincide with the groupings found in the LeuRS tree, revealing that LeuRS evolved independently of its cognate tRNA. Conclusions The study of leucyl-tRNA synthetase in haloarchaea illustrates the importance of gene transfer originating in lineages that went extinct since the transfer occurred. The haloarchaeal LeuRS and tRNALeu did not co-evolve. PMID:22694720

  8. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

    PubMed Central

    Lam, H M; Peng, S S; Coruzzi, G M

    1994-01-01

    Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen. PMID:7846154

  9. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  10. Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing*

    PubMed Central

    Cvetesic, Nevena; Dulic, Morana; Bilus, Mirna; Sostaric, Nikolina; Lenhard, Boris; Gruic-Sovulj, Ita

    2016-01-01

    Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 103-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability. PMID:26921320

  11. Molecular dynamics perspective on the protein thermal stability: a case study using SAICAR synthetase.

    PubMed

    Manjunath, Kavyashree; Sekar, Kanagaraj

    2013-09-23

    The enzyme SAICAR synthetase ligates aspartate with CAIR (5'-phosphoribosyl-4-carboxy-5-aminoimidazole) forming SAICAR (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide) in the presence of ATP. In continuation with our previous study on the thermostability of this enzyme in hyper-/thermophiles based on the structural aspects, here, we present the dynamic aspects that differentiate the mesophilic (E. coli, E. chaffeensis), thermophilic (G. kaustophilus), and hyperthermophilic (M. jannaschii, P. horikoshii) SAICAR synthetases by carrying out a total of 11 simulations. The five functional dimers from the above organisms were simulated using molecular dynamics for a period of 50 ns each at 300 K, 363 K, and an additional simulation at 333 K for the thermophilic protein. The basic features like root-mean-square deviations, root-mean-square fluctuations, surface accessibility, and radius of gyration revealed the instability of mesophiles at 363 K. Mean square displacements establish the reduced flexibility of hyper-/thermophiles at all temperatures. At the simulations time scale considered here, the long-distance networks are considerably affected in mesophilic structures at 363 K. In mesophiles, a comparatively higher number of short-lived (having less percent existence time) Cα, hydrogen bonds, hydrophobic interactions are formed, and long-lived (with higher percentage existence time) contacts are lost. The number of time-averaged salt-bridges is at least 2-fold higher in hyperthermophiles at 363 K. The change in surface accessibility of salt-bridges at 363 K from 300 K is nearly doubled in mesophilic protein compared to proteins from other temperature classes.

  12. Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

    PubMed

    van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

    2015-06-01

    Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex.

  13. Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

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

    1987-06-30

    Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate, and the product was characterized by /sup 31/P, /sup 1/H, and /sup 13/C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by /sup 31/P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 /sup 0/C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by /sup 18/O incorporation from H/sub 2//sup 18/O into P/sub i/, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k/sub cat/ values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.

  14. Cytoplasmic Leucyl-Trna Synthetase of Neurospora Crassa Is Not Specified by the Leu-5 Locus

    PubMed Central

    Benarous, R.; Chow, C. M.; RajBhandary, U. L.

    1988-01-01

    We generated a λgt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, λNCLRSC1 and λNCLRSC2, were obtained which have inserts of ~2 kbp and ~1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that λNCLRSC1 and λNCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by λNCLRSC1 or λNCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an ~115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is ~3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located. PMID:2842224

  15. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    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 CSASs 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 characterized 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 Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) 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 co-ordinating 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.

  16. The mRNA of human cytoplasmic arginyl-tRNA synthetase recruits prokaryotic ribosomes independently.

    PubMed

    Yang, Fang; Ji, Quan-Quan; Ruan, Liang-Liang; Ye, Qing; Wang, En-Duo

    2014-07-25

    There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.

  17. Succinyl-CoA Synthetase: New Antigen Candidate of Bartonella bacilliformis

    PubMed Central

    Gomes, Cláudia; Palma, Noemí; Pons, Maria J.; Magallón-Tejada, Ariel; Sandoval, Isabel; Tinco-Valdez, Carmen; Gutarra, Carlos; del Valle-Mendoza, Juana; Ruiz, Joaquim; Matsuoka, Mayumi

    2016-01-01

    Background Bartonella bacilliformis is the causative agent of Carrion’s disease, a neglected illness with mortality rates of 40–85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas. Methodology/Principal Findings Blood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion’s disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit β (SCS-β). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-β interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31). Conclusions/Significance RT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion’s disease and identify asymptomatic carriers. PMID:27627803

  18. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

    Uppalapati, Maruti; Huang, Ying-Ming; Jackson, Thomas N; Hancock, William O

    2008-09-01

    The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

  19. 21 CFR 886.1630 - AC-powered photostimulator.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false AC-powered photostimulator. 886.1630 Section 886...) MEDICAL DEVICES OPHTHALMIC DEVICES Diagnostic Devices § 886.1630 AC-powered photostimulator. (a) Identification. An AC-powered photostimulator is an AC-powered device intended to provide light stimulus...

  20. 21 CFR 886.1630 - AC-powered photostimulator.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false AC-powered photostimulator. 886.1630 Section 886...) MEDICAL DEVICES OPHTHALMIC DEVICES Diagnostic Devices § 886.1630 AC-powered photostimulator. (a) Identification. An AC-powered photostimulator is an AC-powered device intended to provide light stimulus...

  1. 21 CFR 886.1850 - AC-powered slitlamp biomicroscope.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false AC-powered slitlamp biomicroscope. 886.1850... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Diagnostic Devices § 886.1850 AC-powered slitlamp biomicroscope. (a) Identification. An AC-powered slitlamp biomicroscope is an AC-powered device that is...

  2. 21 CFR 886.1850 - AC-powered slitlamp biomicroscope.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false AC-powered slitlamp biomicroscope. 886.1850... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Diagnostic Devices § 886.1850 AC-powered slitlamp biomicroscope. (a) Identification. An AC-powered slitlamp biomicroscope is an AC-powered device that is...

  3. 21 CFR 888.1240 - AC-powered dynamometer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false AC-powered dynamometer. 888.1240 Section 888.1240...) MEDICAL DEVICES ORTHOPEDIC DEVICES Diagnostic Devices § 888.1240 AC-powered dynamometer. (a) Identification. An AC-powered dynamometer is an AC-powered device intended for medical purposes to...

  4. 21 CFR 886.4440 - AC-powered magnet.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false AC-powered magnet. 886.4440 Section 886.4440 Food... DEVICES OPHTHALMIC DEVICES Surgical Devices § 886.4440 AC-powered magnet. (a) Identification. An AC-powered magnet is an AC-powered device that generates a magnetic field intended to find and...

  5. 21 CFR 886.4440 - AC-powered magnet.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false AC-powered magnet. 886.4440 Section 886.4440 Food... DEVICES OPHTHALMIC DEVICES Surgical Devices § 886.4440 AC-powered magnet. (a) Identification. An AC-powered magnet is an AC-powered device that generates a magnetic field intended to find and...

  6. 21 CFR 886.4440 - AC-powered magnet.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false AC-powered magnet. 886.4440 Section 886.4440 Food... DEVICES OPHTHALMIC DEVICES Surgical Devices § 886.4440 AC-powered magnet. (a) Identification. An AC-powered magnet is an AC-powered device that generates a magnetic field intended to find and...

  7. 21 CFR 886.4440 - AC-powered magnet.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false AC-powered magnet. 886.4440 Section 886.4440 Food... DEVICES OPHTHALMIC DEVICES Surgical Devices § 886.4440 AC-powered magnet. (a) Identification. An AC-powered magnet is an AC-powered device that generates a magnetic field intended to find and...

  8. 21 CFR 886.4440 - AC-powered magnet.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false AC-powered magnet. 886.4440 Section 886.4440 Food... DEVICES OPHTHALMIC DEVICES Surgical Devices § 886.4440 AC-powered magnet. (a) Identification. An AC-powered magnet is an AC-powered device that generates a magnetic field intended to find and...

  9. 21 CFR 888.1240 - AC-powered dynamometer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false AC-powered dynamometer. 888.1240 Section 888.1240...) MEDICAL DEVICES ORTHOPEDIC DEVICES Diagnostic Devices § 888.1240 AC-powered dynamometer. (a) Identification. An AC-powered dynamometer is an AC-powered device intended for medical purposes to...

  10. Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

    PubMed Central

    Tiboni, O; Cammarano, P; Sanangelantoni, A M

    1993-01-01

    The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained. PMID:8098326

  11. AC Electrokinetics of Physiological Fluids for Biomedical Applications.

    PubMed

    Lu, Yi; Liu, Tingting; Lamanda, Ariana C; Sin, Mandy L Y; Gau, Vincent; Liao, Joseph C; Wong, Pak Kin

    2015-12-01

    Alternating current (AC) electrokinetics is a collection of processes for manipulating bulk fluid mass and embedded objects with AC electric fields. The ability of AC electrokinetics to implement the major microfluidic operations, such as pumping, mixing, concentration, and separation, makes it possible to develop integrated systems for clinical diagnostics in nontraditional health care settings. The high conductivity of physiological fluids presents new challenges and opportunities for AC electrokinetics-based diagnostic systems. In this review, AC electrokinetic phenomena in conductive physiological fluids are described followed by a review of the basic microfluidic operations and the recent biomedical applications of AC electrokinetics. The future prospects of AC electrokinetics for clinical diagnostics are presented.

  12. AC Electrokinetics of Physiological Fluids for Biomedical Applications

    PubMed Central

    Lu, Yi; Liu, Tingting; Lamanda, Ariana C.; Sin, Mandy L Y; Gau, Vincent; Liao, Joseph C.; Wong, Pak Kin

    2016-01-01

    AC electrokinetics is a collection of processes for manipulating bulk fluid mass and embedded objects with AC electric fields. The ability of AC electrokinetics to implement the major microfluidic operations, such as pumping, mixing, concentration and separation, makes it possible to develop integrated systems for clinical diagnostics in non-traditional healthcare settings. The high conductivity of physiological fluids presents new challenges and opportunities for AC electrokinetics based diagnostic systems. In this review, AC electrokinetic phenomena in conductive physiological fluids are described followed by a review of the basic microfluidic operations and the recent biomedical applications of AC electrokinetics. The future prospects of AC electrokinetics for clinical diagnostics are presented. PMID:25487557

  13. The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

    PubMed Central

    Berthet-Colominas, C; Seignovert, L; Härtlein, M; Grotli, M; Cusack, S; Leberman, R

    1998-01-01

    The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor. PMID:9582288

  14. Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes 1

    PubMed Central

    Andrews, Jaen; Keegstra, Kenneth

    1983-01-01

    Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum. PMID:16663076

  15. 78 FR 49318 - Availability of Draft Advisory Circular (AC) 90-106A and AC 20-167A

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-13

    ... Federal Aviation Administration Availability of Draft Advisory Circular (AC) 90-106A and AC 20- 167A...: This notice announces the availability of draft Advisory Circular (AC) 90-106A, Enhanced Flight Vision Systems and draft AC 20- 167A, Airworthiness Approval of Enhanced Vision System, Synthetic Vision...

  16. Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

    PubMed

    Anderson, P M

    1989-07-15

    The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

  17. 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.

  18. 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

  19. Design and synthesis of 225Ac radioimmunopharmaceuticals.

    PubMed

    McDevitt, Michael R; Ma, Dangshe; Simon, Jim; Frank, R Keith; Scheinberg, David A

    2002-12-01

    The alpha-particle-emitting radionuclides 213Bi, 211At, 224Ra are under investigation for the treatment of leukemias, gliomas, and ankylosing spondylitis, respectively. 213Bi and 211At were attached to monoclonal antibodies and used as targeted immunotherapeutic agents while unconjugated 224Ra chloride selectively seeks bone. 225Ac possesses favorable physical properties for radioimmunotherapy (10d half-life and 4 net alpha particles), but has a history of unfavorable radiolabeling chemistry and poor metal-chelate stability. We selected functionalized derivatives of DOTA as the most promising to pursue from out of a group of potential 225Ac chelate compounds. A two-step synthetic process employing either MeO-DOTA-NCS or 2B-DOTA-NCS as the chelating moiety was developed to attach 225Ac to monoclonal antibodies. This method was tested using several different IgG systems. The chelation reaction yield in the first step was 93+/-8% radiochemically pure (n=26). The second step yielded 225Ac-DOTA-IgG constructs that were 95+/-5% radiochemically pure (n=27) and the mean percent immunoreactivity ranged from 25% to 81%, depending on the antibody used. This process has yielded several potential novel targeted 225Ac-labeled immunotherapeutic agents that may now be evaluated in appropriate model systems and ultimately in humans.

  20. From Beamline to Scanner with 225Ac

    NASA Astrophysics Data System (ADS)

    Robertson, Andrew K. H.; Ramogida, Caterina F.; Kunz, Peter; Rodriguez-Rodriguez, Cristina; Schaffer, Paul; Sossi, Vesna

    2016-09-01

    Due to the high linear energy transfer and short range of alpha-radiation, targeted radiation therapy using alpha-emitting pharmaceuticals that successfully target small disease clusters will kill target cells with limited harm to healthy tissue, potentially treating the most aggressive forms of cancer. As the parent of a decay chain with four alpha- and two beta-decays, 225Ac is a promising candidate for such a treatment. However, this requires retention of the entire decay chain at the target site, preventing the creation of freely circulating alpha-emitters that reduce therapeutic effect and increase toxicity to non-target tissues. Two major challenges to 225Ac pharmaceutical development exist: insufficient global supply, and the difficulty of preventing toxicity by retaining the entire decay chain at the target site. While TRIUMF works towards large-scale (C i amounts) production of 225Ac, we already use our Isotope Separation On-Line facility to provide small (< 1 mCi) quantities for in-house chemistry and imaging research that aims to improve and assess 225Ac radiopharmaceutical targeting. This presentation provides an overview of this research program and the journey of 225Ac from the beamline to the scanner. This research is funded by the Natural Sciences and Engineering Research Council of Canada.

  1. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

    PubMed

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-09

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.

  2. Altered alpha subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichis coli.

    PubMed

    Hennecke, H; Böck, A

    1975-07-01

    Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenyalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The alpha and beta subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant alpha subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation.

  3. In silico analysis of nonribosomal peptide synthetases of Xanthomonas axonopodis pv. citri: identification of putative siderophore and lipopeptide biosynthetic genes.

    PubMed

    Etchegaray, Augusto; Silva-Stenico, Maria E; Moon, David H; Tsai, Siu M

    2004-01-01

    The genomes of the plant pathogens Xanthomonas axonopodis (Xac) and Xanthomonas campestris (Xcc) were analysed with the aim of deducing their ability to produce nonribosomal peptides. Nonribosomal peptide synthetase (NRPS) genes were identified in two separate loci of Xac. While the genes of locus 1 are common to both strains, locus 2 was only found in Xac. Dissection and phylogenetic analysis of the condensation and thioesterase domains of the NRPSs of loci 1 and 2 of Xac revealed homology, respectively, with siderophore and lipopeptide synthetases. Further analysis of locus 1 revealed genes related to polyketide and polyamine biosynthesis that could be involved in the assembly of substrates for siderophore biosynthesis in both strains. In vitro production of siderophores by both Xac and Xcc was confirmed. Since bacterial siderophores and lipopeptides can be pathogenic and are typically produced nonribosomally, these results suggest that the identified genes could be involved in phytotoxin production.

  4. Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans

    PubMed Central

    Blundell, Ross D.; Williams, Simon J.; Morrow, Carl A.; Ericsson, Daniel J.; Kobe, Bostjan; Fraser, James A.

    2013-01-01

    With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P212121 and diffracted to 2.2 Å resolution. PMID:23989157

  5. Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans.

    PubMed

    Blundell, Ross D; Williams, Simon J; Morrow, Carl A; Ericsson, Daniel J; Kobe, Bostjan; Fraser, James A

    2013-09-01

    With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 Å resolution.

  6. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-01

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  7. Severe respiratory failure as a presenting feature of an interstitial lung disease associated with anti-synthetase syndrome (ASS).

    PubMed

    Piroddi, Ines Maria Grazia; Ferraioli, Gianluca; Barlascini, Cornelius; Castagneto, Corrado; Nicolini, Antonello

    2016-07-01

    Anti-synthetase syndrome (ASS) is defined as a heterogeneous connective tissue disorder characterized by the association of an interstitial lung disease (ILD) with or without inflammatory myositis with the presence of anti-aminoacyl-tRNA-synthetase antibodies. ILD is one of the major extra-muscular manifestations of polymyositis and dermatomyositis. We report a case of a patient with dyspnea, cough, and intermittent fever as well as ILD associated ASS in the absence of muscular involvement. This patient was admitted to the emergency department with severe respiratory failure requiring non-invasive ventilation. Our patient's case demonstrates that the diagnosis of ASS may not be obvious. However, its diagnosis leads to appropriate and potentially life-saving treatment.

  8. Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

    PubMed Central

    Sauter, Claude; Lorber, Bernard; Gaudry, Agnès; Karim, Loukmane; Schwenzer, Hagen; Wien, Frank; Roblin, Pierre; Florentz, Catherine; Sissler, Marie

    2015-01-01

    Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation. PMID:26620921

  9. [Glutathion-synthetase deficiency with 5-oxoprolinuria. Two new cases and a review of the literature (author's transl)].

    PubMed

    Boivin, P; Galand, C; Schaison, G

    1978-05-06

    Hereditary deficiency in glutathion- synthetase is a rare disease presenting up to now either with a congenital non-spherocytic anaemia or with a metabolic acidosis, most often neonatal and accompanied by a pyroglutamic amino-aciduria (5-oxoprolinuria). These two syndrome may be present together or exist independently. Pyroglutamic amino-aciduria is the result of extension of the deficiency to non-haematopoietic cells, in particular renal. Two new cases of glutathion-synthetase deficiency are reported: both with haemolytic anaemia and moderate pyroglutamic amino-aciduria, in the absence of clinical signs of metabolic acidosis. The clinical, haematological and biochemical heterogeneity of the deficiency is illustrated by these two cases and datas from the literature.

  10. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    SciTech Connect

    Timofeev, V. I. E-mail: tostars@mail.ru; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-15

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  11. The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

    PubMed

    Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code.

  12. A Previously Unknown Oxalyl-CoA Synthetase Is Important for Oxalate Catabolism in Arabidopsis[W

    PubMed Central

    Foster, Justin; Kim, Hyun Uk; Nakata, Paul A.; Browse, John

    2012-01-01

    Oxalate is produced by several catabolic pathways in plants. The best characterized pathway for subsequent oxalate degradation is via oxalate oxidase, but some species, such as Arabidopsis thaliana, have no oxalate oxidase activity. Previously, an alternative pathway was proposed in which oxalyl-CoA synthetase (EC 6.2.1.8) catalyzes the first step, but no gene encoding this function has been found. Here, we identify ACYL-ACTIVATING ENZYME3 (AAE3; At3g48990) from Arabidopsis as a gene encoding oxalyl-CoA synthetase. Recombinant AAE3 protein has high activity against oxalate, with Km = 149.0 ± 12.7 μM and Vmax = 11.4 ± 1.0 μmol/min/mg protein, but no detectable activity against other organic acids tested. Allelic aae3 mutants lacked oxalyl-CoA synthetase activity and were unable to degrade oxalate into CO2. Seeds of mutants accumulated oxalate to levels threefold higher than the wild type, resulting in the formation of oxalate crystals. Crystal formation was associated with seed coat defects and substantially reduced germination of mutant seeds. Leaves of mutants were damaged by exogenous oxalate and more susceptible than the wild type to infection by the fungus Sclerotinia sclerotiorum, which produces oxalate as a phytotoxin to aid infection. Our results demonstrate that, in Arabidopsis, oxalyl-CoA synthetase encoded by AAE3 is required for oxalate degradation, for normal seed development, and for defense against an oxalate-producing fungal pathogen. PMID:22447686

  13. Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display.

    PubMed

    Zou, Yekui; Yin, Jun

    2008-10-15

    To engineer the substrate specificities of nonribosomal peptide synthetases (NRPS), we developed a method to display NRPS modules on M13 phages and select catalytically active adenylation (A) domains that would load azide functionalized substrate analogs to the neighboring peptidyl carrier protein (PCP) domains. Biotin conjugated difluorinated cyclooctyne was used for copper free cycloaddition with an azide substituted substrate attached to PCP. Biotin-labeled phages were selected by binding to streptavidin.

  14. Effect of membrane-associated f1 bacteriophage coat protein upon the activity of Escherichia coli phosphatidylserine synthetase.

    PubMed Central

    Chamberlain, B K; Webster, R E

    1978-01-01

    The effects of insertion of the major coat protein of f1 bacteriophage into Escherichia coli membranes were investigated under conditions allowing in vivo analysis of phosphatidylserine synthesis. An E. coli strain possessing a temperature-sensitive phosphatidylserine decarboxylase was utilized under conditions in which the decarboxylase activity was reduced but nonlethal. The presence of the coat protein in the host membranes inhibits the activity of the phosphatidylserine synthetase and perhaps affects the activity of the phosphatidylserine decarboxylase. PMID:211116

  15. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    PubMed Central

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O.

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

  16. Nanomolar inhibitors of Mycobacterium tuberculosis glutamine synthetase 1: synthesis, biological evaluation and X-ray crystallographic studies.

    PubMed

    Couturier, Cédric; Silve, Sandra; Morales, Renaud; Pessegue, Bernard; Llopart, Sylvie; Nair, Anil; Bauer, Armin; Scheiper, Bodo; Pöverlein, Christoph; Ganzhorn, Axel; Lagrange, Sophie; Bacqué, Eric

    2015-04-01

    A series of imidazo[1,2-a]indeno[1,2-e]pyrazin-4-ones that potently inhibit M. tuberculosis glutamine synthetase (GlnA1) has been identified by high throughput screening. Exploration of this series was performed owing to a short chemistry program. Despite possibly nanomolar inhibitions, none of these compounds was active on whole cell Mtb, suggesting that GlnA1 may not be a suitable target to find new anti-tubercular drugs.

  17. Numerical simulation of ac plasma arc thermodynamics

    NASA Astrophysics Data System (ADS)

    Wu, Han-Ming; Carey, G. F.; Oakes, M. E.

    1994-05-01

    A mathematical model and approximate analysis for the energy distribution of an ac plasma arc with a moving boundary is developed. A simplified electrical conductivity function is assumed so that the dynamic behavior of the arc may be determined, independent of the gas type. The model leads to a reduced set of non-linear partial differential equations which governs the quasi-steady ac arc. This system is solved numerically and it is found that convection plays an important role, not only in the temperature distribution, but also in arc disruptions. Moreover, disruptions are found to be influenced by convection only for a limited frequency range. The results of the present studies are applicable to the frequnecy range of 10-10(exp 2) Hz which includes most industry ac arc frequencies.

  18. Numerical Simulation of AC Plasma Arc Thermodynamics

    NASA Astrophysics Data System (ADS)

    Wu, Han-Ming; Carey, G. F.; Oakes, M. E.

    1994-05-01

    A mathematical model and approximate analysis for the energy distribution of an ac plasma arc with a moving boundary is developed. A simplified electrical conductivity function is assumed so that the dynamic behavior of the arc may be determined, independent of the gas type. The model leads to a reduced set of non-linear partial differential equations which governs the quasi-steady ac arc. This system is solved numerically and it is found that convection plays an important role, not only in the temperature distribution, but also in arc disruptions. Moreover, disruptions are found to be influenced by convection only for a limited frequency range. The results of the present studies are applicable to the frequency range of 10-102 Hz which includes most industry ac arc frequencies.

  19. Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

    NASA Astrophysics Data System (ADS)

    Abramchik, Yu. A.; Timofeev, V. I.; Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S.; Kuranova, I. P.

    2017-01-01

    Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus ( T. th HB27) has high thermal stability and shows maximum activity at 75°C, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

  20. The Drosophila ebony gene is closely related to microbial peptide synthetases and shows specific cuticle and nervous system expression.

    PubMed

    Hovemann, B T; Ryseck, R P; Walldorf, U; Störtkuhl, K F; Dietzel, I D; Dessen, E

    1998-10-09

    The previously detected ebony (e) locus (Caizzi et al., 1987) consists of a complex gene structure that is divided into seven exons. An open reading frame encoding the putative Ebony protein of 98.5 kDa exhibits homology to a family of peptide synthetases (Stachelhaus and Marahiel, 1995), in good correlation with the proposed function as beta-alanyl-dopamine synthetase. Multiple ebony transcripts are detected throughout development. P-factor mediated transformation of genomic DNA rescues the cuticle, electrophysiological and behavioural phenotypes. Fusion of the ebony reading frame with that of beta-galactosidase of E. coli reveals expression in cuticle and nervous system. Strong staining in the first and, to a lesser extent, in the second optic neuropile may reflect the pronounced visual defect observed in ebony mutants. In addition, weak central brain and thoracic ganglion expression is detected in flies. Conservation of a multidomain protein structure known from peptide synthetases should have functional implications on the putative reaction mechanism of peptide bond formation.

  1. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

    SciTech Connect

    Guo, Min; Ignatov, Michael; Musier-Forsyth, Karin; Schimmel, Paul; Yang, Xiang-Lei

    2008-09-17

    In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The {alpha}{sub 2} homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that {alpha}{sub 2} LysRS is tightly bound into the core of the MSC with homodimeric {beta}{sub 2} p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive {alpha}{sub 2}{beta}{sub 2} heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-{angstrom} crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryote-specific sequences, one in the catalytic- and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein-protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.

  2. Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.

    PubMed Central

    Williamson, R M; Oxender, D L

    1990-01-01

    A role for the leucyl-tRNA synthetase (EC 6.1.1.4) has been established for regulating the transport of leucine across the inner membrane of Escherichia coli by the leucine, isoleucine, valine (LIV-I) transport system. This transport system is mediated by interactions of periplasmic binding proteins with a complex of membrane-associated proteins, and transcription of the high-affinity branched-chain amino acid transport system genes is repressed by growth of E. coli on high levels of leucine. We now report results from sequence comparisons and structural modeling studies, which indicate that the leucine-specific binding protein, one of the periplasmic components of the LIV-I transport system, contains a 121-residue stretch, representing 36% of the mature protein, which displays both sequence and structural similarities to a region within the putative nucleotide-binding domain of leucyl-tRNA synthetase. Early fusion events between ancestral genes for the leucine-specific binding protein and leucyl-tRNA synthetase could account for the similarity and suggest that processes of aminoacylation and transport for leucine in E. coli may be performed by evolutionarily interrelated proteins. PMID:2191293

  3. Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

    PubMed

    Naggert, J; Witkowski, A; Mikkelsen, J; Smith, S

    1988-01-25

    A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

  4. MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

    PubMed Central

    Li, Rongzhong; Macnamara, Lindsay M.; Leuchter, Jessica D.; Alexander, Rebecca W.; Cho, Samuel S.

    2015-01-01

    While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD) simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes. PMID:26184179

  5. Crystal structures of three protozoan homologs of tryptophanyl-tRNA synthetase.

    PubMed

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, Robert; Napuli, Alberto J; Kim, Jessica E; Buckner, Frederick S; Van Voorhis, Wesley C; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Hol, Wim G J

    2011-05-01

    Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNA(Trp) molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNA(Trp) after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNA(Trp) recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual Trp

  6. Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

    PubMed Central

    Janata, Jiri; Kadlcik, Stanislav; Koberska, Marketa; Ulanova, Dana; Kamenik, Zdenek; Novak, Petr; Kopecky, Jan; Novotna, Jitka; Radojevic, Bojana; Plhackova, Kamila; Gazak, Radek; Najmanova, Lucie

    2015-01-01

    In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system

  7. Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement.

    PubMed

    Zhang, Shaowei; Han, Yingkun; Kumar, Ashok; Gao, Haofeng; Liu, Ziduo; Hu, Nan

    2017-02-07

    A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

  8. Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

    SciTech Connect

    Cruzen, M.E.

    1993-01-01

    Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closely related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.

  9. Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.

    PubMed

    Wei, Na; Shi, Yi; Truong, Lan N; Fisch, Kathleen M; Xu, Tao; Gardiner, Elisabeth; Fu, Guangsen; Hsu, Yun-Shiuan Olivia; Kishi, Shuji; Su, Andrew I; Wu, Xiaohua; Yang, Xiang-Lei

    2014-10-23

    Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage.

  10. Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

    PubMed Central

    Park, Hyun Bong; Perez, Corey E; Barber, Karl W; Rinehart, Jesse; Crawford, Jason M

    2017-01-01

    Nonribosomal peptides represent a large class of metabolites with pharmaceutical relevance. Pteridines, such as pterins, folates, and flavins, are heterocyclic metabolites that often serve as redox-active cofactors. The biosynthetic machineries for construction of these distinct classes of small molecules operate independently in the cell. Here, we discovered an unprecedented nonribosomal peptide synthetase-like-pteridine synthase hybrid biosynthetic gene cluster in Photorhabdus luminescens using genome synteny analysis. P. luminescens is a Gammaproteobacterium that undergoes phenotypic variation and can have both pathogenic and mutualistic roles. Through extensive gene deletion, pathway-targeted molecular networking, quantitative proteomic analysis, and NMR, we show that the genetic locus affects the regulation of quorum sensing and secondary metabolic enzymes and encodes new pteridine metabolites functionalized with cis-amide acyl-side chains, termed pepteridine A (1) and B (2). The pepteridines are produced in the pathogenic phenotypic variant and represent the first reported metabolites to be synthesized by a hybrid NRPS-pteridine pathway. These studies expand our view of the combinatorial biosynthetic potential available in bacteria. DOI: http://dx.doi.org/10.7554/eLife.25229.001

  11. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    SciTech Connect

    Timofeev, V. I. Abramchik, Yu. A. Zhukhlistova, N. E. Kuranova, I. P.

    2015-09-15

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6{sub 3}22 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  12. The Effect of Nitrogen Nutrition on the Cellular Localization of Glutamine Synthetase Isoforms in Barley Roots.

    PubMed Central

    Peat, L. J.; Tobin, A. K.

    1996-01-01

    Glutamine synthetase (GS) was detected by immunogold localization in the cytosol and plastids of roots of 7-d-old barley (Hordeum vulgare L. cv Klaxon) seedlings grown in the presence or absence of NO3- (15 mM) or NH4+ (30 mM). The number of GS polypeptides changed during root development, and this was affected by N nutrition. There was no evidence of a NO3--inducible root plastid GS.In apical 5- to 10-mm regions of the root the concentration of immunogold labeling of cytosolic GS was higher in the cortical parenchyma than in the vascular cells of the stele, irrespective of N nutrition. This labeling was at least 50% higher in both cell types in N-free compared with N-grown (either NO3- or NH4+) seedlings. In contrast, GS specific activity was highest in roots of NO3--grown seedlings. It is suggested that this indicates the presence of inactive GS in roots grown without N. This study has identified both cell- and development-specific responses of GS to N nutrition. PMID:12226350

  13. Overexpression of a glutamine synthetase gene affects growth and development in sorghum.

    PubMed

    Urriola, Jazmina; Rathore, Keerti S

    2015-06-01

    Nitrogen is a primary macronutrient in plants, and nitrogen fertilizers play a critical role in crop production and yield. In this study, we investigated the effects of overexpressing a glutamine synthetase (GS) gene on nitrogen metabolism, and plant growth and development in sorghum (Sorghum bicolor L., Moench). GS catalyzes the ATP dependent reaction between ammonia and glutamate to produce glutamine. A 1,071 bp long coding sequence of a sorghum cytosolic GS gene (Gln1) under the control of the maize ubiquitin (Ubq) promoter was introduced into sorghum immature embryos by Agrobacterium-mediated transformation. Progeny of the transformants exhibited higher accumulation of the Gln1 transcripts and up to 2.2-fold higher GS activity compared to the non-transgenic controls. When grown under optimal nitrogen conditions, these Gln1 transgenic lines showed greater tillering and up to 2.1-fold increase in shoot vegetative biomass. Interestingly, even under greenhouse conditions, we observed a seasonal component to both these parameters and the grain yield. Our results, showing that the growth and development of sorghum Gln1 transformants are also affected by N availability and other environmental factors, suggest complexity of the relationship between GS activity and plant growth and development. A better understanding of other control points and the ability to manipulate these will be needed to utilize the transgenic technology to improve nitrogen use efficiency of crop plants.

  14. Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

    PubMed Central

    Tsai, F Y; Coruzzi, G M

    1990-01-01

    Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1968003

  15. Chitin synthetases in Candida albicans: a review on their subcellular distribution and biological function.

    PubMed

    Martínez, J P; Gozalbo, D

    1994-09-01

    In the light of recent genetic advances, some results regarding chitin biosynthetic activities are reviewed in this paper. Genes coding for distinct enzymes displaying chitin synthetase activities have been characterized in Saccharomyces cerevisiae as well as in other fungal species including Candida albicans. Several activities seem to exist in the cells: (i) one zymogenic, located in cytoplasmic vesicles called chitosomes, although the presence of other types of vesicles with zymogenic activity cannot be completely discarded, and (ii) plasma membrane associated activities (the active enzyme and probably two distinct pools of zymogenic activity). Possible relationships between these activities, if any, remain to be determined. These multiplicity of enzymes is not surprising taking into account that chitin biosynthesis is required during very well defined temporal and spatial events of the cell cycle. A general repair function for one of the chitin biosynthetic activities is proposed as a possible salvage mechanism to warrant cell survival after wall damage has been caused, since chitin appears to be the most suitable polymer to carry out this function due to its particular physico-chemical properties.

  16. Brugia malayi Asparaginyl - tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis

    PubMed Central

    D, Jeeva Jothi; Dhanraj, Muthu; Solaiappan, Shanmugam; Sivanesan, Sanjana; Kron, Michael; Dhanasekaran, Anuradha

    2016-01-01

    A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis. PMID:26751209

  17. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

    PubMed

    Rydzak, Thomas; Garcia, David; Stevenson, David M; Sladek, Margaret; Klingeman, Dawn M; Holwerda, Evert K; Amador-Noguez, Daniel; Brown, Steven D; Guss, Adam M

    2017-04-08

    Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

  18. A unique insertion in the CP1 domain of Giardia lamblia leucyl-tRNA synthetase.

    PubMed

    Zhou, Xiao-Long; Yao, Peng; Ruan, Liang-Liang; Zhu, Bin; Luo, Jun; Qu, Liang-Hu; Wang, En-Duo

    2009-02-17

    Leucyl-tRNA synthetase (LeuRS) catalyzes the esterification of the tRNA(Leu) isoacceptor with leucine. It contains a large insertion domain, connective peptide 1 (CP1), for amino acid editing. Here, we cloned the gene encoding LeuRS from Giardia lamblia (GlLeuRS), one of the most ancient eukaryotes. GlLeuRS was purified from an Escherichia coli overproduction strain, and its properties were investigated. The isolated CP1 domain of GlLeuRS (GlLeuRS-CP1) was an active protein for editing mischarged G. lamblia tRNA(Leu)(AAG) (GltRNA(Leu)). Insertion of 49 amino acid residues within the CP1 domain (the so-called 49-amino acid motif) was important for the optimal aminoacylation activity of GlLeuRS and was crucial for the editing capacity of GlLeuRS-CP1. Additionally, the motif can confer editing activity on the editing-defective isolated CP1 domain from E. coli LeuRS (EcLeuRS-CP1). We also found that GlLeuRS could not rescue a Saccharomyces cerevisiae leuS null strain, suggesting different recognition modes for these two LeuRSs with respect to tRNA(Leu).

  19. Structural characterization of antibiotic self-immunity tRNA synthetase in plant tumour biocontrol agent

    PubMed Central

    Chopra, Shaileja; Palencia, Andrés; Virus, Cornelia; Schulwitz, Sarah; Temple, Brenda R.; Cusack, Stephen; Reader, John

    2016-01-01

    Antibiotic-producing microbes evolved self-resistance mechanisms to avoid suicide. The biocontrol Agrobacterium radiobacter K84 secretes the Trojan Horse antibiotic agrocin 84 that is selectively transported into the plant pathogen A. tumefaciens and processed into the toxin TM84. We previously showed that TM84 employs a unique tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase (LeuRS), while the TM84-producer prevents self-poisoning by expressing a resistant LeuRS AgnB2. We now identify a mechanism by which the antibiotic-producing microbe resists its own toxin. Using a combination of structural, biochemical and biophysical approaches, we show that AgnB2 evolved structural changes so as to resist the antibiotic by eliminating the tRNA-dependence of TM84 binding. Mutagenesis of key resistance determinants results in mutants adopting an antibiotic-sensitive phenotype. This study illuminates the evolution of resistance in self-immunity genes and provides mechanistic insights into a fascinating tRNA-dependent antibiotic with applications for the development of anti-infectives and the prevention of biocontrol emasculation. PMID:27713402

  20. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content

    PubMed Central

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L.; Paradiso, Annalisa; Gu, Yong Q.; Blanco, Antonio; de Pinto, Maria C.; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  1. Physiological Studies of Glutamine Synthetases I and III from Synechococcus sp. WH7803 Reveal Differential Regulation

    PubMed Central

    Domínguez-Martín, María Agustina; Díez, Jesús; García-Fernández, José M.

    2016-01-01

    The marine picocyanobacterium Synechococcus sp. WH7803 possesses two glutamine synthetases (GSs; EC 6.3.1.2), GSI encoded by glnA and GSIII encoded by glnN. This is the first work addressing the physiological regulation of both enzymes in a marine cyanobacterial strain. The increase of GS activity upon nitrogen starvation was similar to that found in other model cyanobacteria. However, an unusual response was found when cells were grown under darkness: the GS activity was unaffected, reflecting adaptation to the environment where they thrive. On the other hand, we found that GSIII did not respond to nitrogen availability, in sharp contrast with the results observed for this enzyme in other cyanobacteria thus far studied. These features suggest that GS activities in Synechococcus sp. WH7803 represent an intermediate step in the evolution of cyanobacteria, in a process of regulatory streamlining where GSI lost the regulation by light, while GSIII lost its responsiveness to nitrogen. This is in good agreement with the phylogeny of Synechococcus sp. WH7803 in the context of the marine cyanobacterial radiation. PMID:27446010

  2. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    PubMed

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

  3. Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

    PubMed

    Fonseca, Maisa S; Comini, Marcelo A; Resende, Bethânia V; Santi, Ana Maria M; Zoboli, Antônio P; Moreira, Douglas S; Murta, Silvane M F

    2017-04-01

    Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (Sb(III))-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in Sb(III)-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of Sb(III) in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to Sb(III) than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to Sb(III), and confirms a role of these genes in the Sb(III)-resistance phenotype.

  4. Glutamine Synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma

    PubMed Central

    Tardito, Saverio; Oudin, Anaïs; Ahmed, Shafiq U.; Fack, Fred; Keunen, Olivier; Zheng, Liang; Miletic, Hrvoje; Sakariassen, Per Øystein; Weinstock, Adam; Wagner, Allon; Lindsay, Susan L.; Hock, Andreas K.; Barnett, Susan C.; Ruppin, Eytan; Mørkve, Svein Harald; Lund-Johansen, Morten; Chalmers, Anthony J.; Bjerkvig, Rolf; Niclou, Simone P.; Gottlieb, Eyal

    2015-01-01

    L-Glutamine (Gln) functions physiologically to balance tissue requirements of carbon and nitrogen. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle and, that inhibiting glutaminolysis does not affect proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by Glutamine Synthetase (GS) (cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, 13C-glucose tracing showed that GS produces Gln from TCA cycle-derived carbons. Finally, while it is contributed only marginally by the circulation, the Gln required for the growth of GBM tumours is either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes. PMID:26595383

  5. Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

    PubMed Central

    Carpenter, E J; Bergman, B; Dawson, R; Siddiqui, P J; Söderbäck, E; Capone, D G

    1992-01-01

    We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation. Images PMID:1359837

  6. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Kuranova, I. P.

    2015-09-01

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6322 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  7. Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli

    SciTech Connect

    Barros, M.E.C.; Rawlings, D.E.; Woods, D.R.

    1985-12-01

    The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/SO/sub 4/ as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.

  8. Production of Non-Ribosomal Peptide Synthetase (NRPS)- Dependent Siderophore by Aeromonas Isolates

    PubMed Central

    Amsaveni, Ramasamy; Sureshkumar, Muthusamy; Aravinth, Arthanari; Mary, Joseph Reshma; Vivekanandhan, Govindasami

    2016-01-01

    Background: Aeromonas species are Gram-negative ubiquitous bacteria, facultative anaerobic rods that infect both invertebrates and vertebrates. Various fish species develop hemorrhagic disease and furunculosis due to Aeromonas spp. Aeromonas strains generate certain active compounds such as siderophores, which are the final products of non-ribosomal peptide synthetase (NRPS) activity. The present study attempted to investigate the prevalence of Aeromonas isolates in marketed fish sources. We also examined the siderophore production ability of these isolates. Methods: Among the molecular tools, 16S rRNA analysis was used to identify Aeromonas species and their epidemiological distributions. The hemolytic activity of the strains and biochemical assays were used to confirm the identity of the isolates. We also determined the chemical nature of siderophores in these strains. Results: A total of seven Aeromonas isolates obtained from fish were included to determine the siderophore production. Of 7 isolates, 4 produced siderophore, and their chemical nature was also determined. The siderophore produced by Aeromonas was invariably found to be of hydroxamate. Four Aeromonas isolates were selected for PCR identification of NRPS-encoding gene. The conserved sequence was present in all four selected isolates. Furthermore, siderophores were qualitatively tested for their antibacterial activity against pathogenic bacteria and a significant level of inhibitory activity was observed in siderophores from the four isolates. Conclusion: Our results showed the ability of the isolated strains in production of siderophores with a high level of activity against Salmonella paratyphi. These siderophores could find applications in biomedical industries. PMID:27155016

  9. Molecular evolution of aminoacyl tRNA synthetase proteins in the early history of life.

    PubMed

    Fournier, Gregory P; Andam, Cheryl P; Alm, Eric J; Gogarten, J Peter

    2011-12-01

    Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.

  10. Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

    NASA Astrophysics Data System (ADS)

    Fournier, Gregory P.; Andam, Cheryl P.; Alm, Eric J.; Gogarten, J. Peter

    2011-12-01

    Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.

  11. Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development.

    PubMed Central

    Uwer, U; Willmitzer, L; Altmann, T

    1998-01-01

    Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment. PMID:9707529

  12. 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.

  13. Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles.

    PubMed

    Ottesen, Elizabeth A; Leadbetter, Jared R

    2011-05-01

    In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.

  14. Farnesyl pyrophosphate synthetase. Mechanistic studies of the 1'-4 coupling reaction with 2-fluorogeranyl pyrophosphate.

    PubMed

    Poulter, C D; Argyle, J C; Mash, E A

    1978-10-25

    The mechanism of the 1'-4 coupling reaction between isopentenyl pyrophosphate and geranyl pyrophosphate catalyzed by farnesyl pyrophosphate synthetase from porcine liver was studied with the allylic substrate analogue 2-fluorogeranyl pyrophosphate. 2-Fluorogeranyl pyrophosphate is an alternate substrate for the enzyme, yielding 6-fluorofarnesyl pyrophosphate upon condensation with isopentenyl pyrophosphate. The Michaelis constant for the fluoroanalogue, Km = 1.1 micron, is similar to that measured for geranyl pyrophosphate, Km = 0.7 micron. However, the rate of condensation with the fluoroanalogue was only 8.4 X 10(-4) that of the normal reaction. A similar rate of depression (4.4 X 10(-3)) was found for solvolysis of geranyl methanesulfonate and the corresponding 2-fluoro derivative, reactions known to proceed via cationic intermediates. In contrast, displacement of chlorine from geranyl chloride and 2-fluorogeranyl chloride by cyanide showed a small (2-fold) rate enhancement for the fluoro compound. Finally, 2-fluorogeranyl pyrophosphate is a competitive inhibitor against geranyl pyrophosphate. These data are interpreted in terms of an ionization-condensation-elimination mechanism for the 1'-4 coupling reaction.

  15. Chemical validation of trypanothione synthetase: a potential drug target for human trypanosomiasis.

    PubMed

    Torrie, Leah S; Wyllie, Susan; Spinks, Daniel; Oza, Sandra L; Thompson, Stephen; Harrison, Justin R; Gilbert, Ian H; Wyatt, Paul G; Fairlamb, Alan H; Frearson, Julie A

    2009-12-25

    In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 x EC(50) for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.

  16. The inactivating factor of glutamine synthetase, IF7, is a "natively unfolded" protein

    PubMed Central

    Muro-Pastor, M. Isabel; Barrera, Francisco N.; Reyes, José C.; Florencio, Francisco J.; Neira, José L.

    2003-01-01

    Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein-protein interactions with a small 65-residue-long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a "natively unfolded" protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant of KD = 0.3 ± 0.1 μM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded. PMID:12824490

  17. Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.

    PubMed

    Zimhony, O; Cox, J S; Welch, J T; Vilchèze, C; Jacobs, W R

    2000-09-01

    Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.

  18. An exposed cysteine residue of human angiostatic mini tryptophanyl-tRNA synthetase.

    PubMed

    Wakasugi, Keisuke

    2010-04-13

    Human tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNA(Trp). Human TrpRS exists in two forms: a major form that is the full-length protein and a truncated form (mini TrpRS) in which most of the N-terminal extension is absent. Human mini, but not full-length, TrpRS has angiostatic activity. Because the full-length protein, which lacks angiostatic activity, has all of the amino acid determinants of the mini form, which has activity, I searched for conformational differences between the two proteins. Using a disulfide cross-linking assay, I showed that the molecular environment around Cys62 is significantly different between the two proteins. This difference can be explained by inspection of the three-dimensional structure of the full-length protein. These results give a clear demonstration of a significant difference, around a specific residue (Cys62), between a potent angiostatic and nonangiostatic version of human TrpRS.

  19. Zinc-mediated amino acid discrimination in cysteinyl-tRNA synthetase.

    PubMed

    Zhang, Chun-Mei; Christian, Thomas; Newberry, Kate J; Perona, John J; Hou, Ya-Ming

    2003-04-11

    Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal-thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc-thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step.

  20. Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants

    SciTech Connect

    Tsai, Fongying; Coruzzi, G. )

    1991-10-01

    Asparagine synthetase (AS) mRNA in Pisum sativum accumulates preferentially in plants grown in the dark. Nuclear run-on experiments demonstrate that expression of both the AS1 and AS2 genes is negatively regulated by light at the level of transcription. A decrease in the transcriptional rate of the AS1 gene can be detected as early as 20 min after exposure to light. Time course experiments reveal that the levels of AS mRNA fluctuate dramatically during a normal light/dark cycle. This is due to a direct effect of light and not to changes associated with circadian rhythm. A novel finding is that the light-repressed expression of the AS1 gene is as dramatic nonphotosynthetic organs such as roots as it is in leaves. Experiments demonstrate that the small amount of light which passes through the soil is sufficient to repress AS1 expression in roots, indicating that light has a direct effect on AS1 gene expression in roots. The negative regulation of AS gene expression by light was shown to be a general phenomenon in plants which also occurs in nonlegumes such as Nicotiana plumbaginifolia and Nicotiana tabacum. Thus, the AS genes can serve as a model with which to dissect the molecular basis for light-regulated transcriptional repression in plants.

  1. Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase

    PubMed Central

    Liu, Ru-Juan; Lukarska, Maria; Gut, Jiri; Bougdour, Alexandre; Touquet, Bastien; Wang, En-Duo; Li, Xianfeng; Alley, M. R. K.; Freund, Yvonne R.; Rosenthal, Philip J.; Hakimi, Mohamed-Ali

    2016-01-01

    The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNALeu in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents. PMID:27431220

  2. Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes.

    PubMed

    Li, Jie; Dong, Jun-De; Yang, Jian; Luo, Xiong-Ming; Zhang, Si

    2014-10-01

    The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed <70% similarity to their closest relatives, which suggested the novelty of these genes. This study helps uncover the genetic capacity of stony coral-associated actinomycetes to produce bioactive molecules.

  3. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.

  4. Phylogenetic Study of Polyketide Synthases and Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Mycotoxins

    PubMed Central

    Gallo, Antonia; Ferrara, Massimo; Perrone, Giancarlo

    2013-01-01

    Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: β-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species. PMID:23604065

  5. No rosetta stone for a sense-antisense origin of aminoacyl tRNA synthetase classes.

    PubMed

    Williams, Tom A; Wolfe, Kenneth H; Fares, Mario A

    2009-02-01

    Aminoacyl tRNA synthetases (aaRS) are crucial enzymes that join amino acids to their cognate tRNAs, thereby implementing the genetic code. These enzymes fall into two unrelated structural classes whose evolution has not been explained. The leading hypothesis, proposed by Rodin and Ohno, is that the two classes originated as a pair of sense-antisense genes encoded on opposite strands of a single DNA molecule. This unusual idea obtained its main support from reports of a "Rosetta stone": a locus where genes for heat shock protein 70 (HSP70) and an Nicotinamide adenine dinulecotide-specific glutamate dehydrogenase (NAD-GDH), which are structurally homologous to the two classes of aaRS, overlap extensively on complementary DNA strands. This remarkable locus was first characterized in the oomycete Achlya klebsiana and has since been reported in many other species. Here we present evidence that the open reading frames on the antisense strand of HSP70 genes are spurious, and we identify a more probable candidate for the gene encoding the oomycete NAD-GDH enzyme. These results cast extensive doubt on the Rosetta Stone argument.

  6. Partial Purification and Characterization of Dihydrodipicolinic Acid Synthetase from Sporulating Bacillus megaterium1

    PubMed Central

    Webster, Francis H.; Lechowich, Richard V.

    1970-01-01

    Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent Km values were 4.6 × 10−4 and 5.0 × 10−4m for β-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The Ea was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the ΔF, ΔH, and ΔS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively. PMID:4983642

  7. The mitochondrial folylpolyglutamate synthetase gene is required for nitrogen utilization during early seedling development in arabidopsis.

    PubMed

    Jiang, Ling; Liu, Yanyan; Sun, Hong; Han, Yueting; Li, Jinglai; Li, Changkun; Guo, Wenzhu; Meng, Hongyan; Li, Sha; Fan, Yunliu; Zhang, Chunyi

    2013-02-01

    Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO(2) partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism.

  8. Human selenophosphate synthetase 1 has five splice variants with unique interactions, subcellular localizations and expression patterns

    SciTech Connect

    Kim, Jin Young; Lee, Kwang Hee; Shim, Myoung Sup; Shin, Hyein; Xu, Xue-Ming; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2010-06-18

    Selenophosphate synthetase 1 (SPS1) is an essential cellular gene in higher eukaryotes. Five alternative splice variants of human SPS1 (major type, {Delta}E2, {Delta}E8, +E9, +E9a) were identified wherein +E9 and +E9a make the same protein. The major type was localized in both the nuclear and plasma membranes, and the others in the cytoplasm. All variants form homodimers, and in addition, the major type forms a heterodimer with {Delta}E2, and {Delta}E8 with +E9. The level of expression of each splice variant was different in various cell lines. The expression of each alternative splice variant was regulated during the cell cycle. The levels of the major type and {Delta}E8 were gradually increased until G2/M phase and then gradually decreased. {Delta}E2 expression peaked at mid-S phase and then gradually decreased. However, +E9/+E9a expression decreased gradually after cell cycle arrest. The possible involvement of SPS1 splice variants in cell cycle regulation is discussed.

  9. Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

    PubMed

    Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

    2016-11-10

    Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation.

  10. Impaired novelty acquisition and synaptic plasticity in congenital hyperammonemia caused by hepatic glutamine synthetase deficiency

    PubMed Central

    Chepkova, Aisa N.; Sergeeva, Olga A.; Görg, Boris; Haas, Helmut L.; Klöcker, Nikolaj; Häussinger, Dieter

    2017-01-01

    Genetic defects in ammonia metabolism can produce irreversible damage of the developing CNS causing an impairment of cognitive and motor functions. We investigated alterations in behavior, synaptic plasticity and gene expression in the hippocampus and dorsal striatum of transgenic mice with systemic hyperammonemia resulting from conditional knockout of hepatic glutamine synthetase (LGS-ko). These mice showed reduced exploratory activity and delayed habituation to a novel environment. Field potential recordings from LGS-ko brain slices revealed significantly reduced magnitude of electrically-induced long-term potentiation (LTP) in both CA3-CA1 hippocampal and corticostriatal synaptic transmission. Corticostriatal but not hippocampal slices from LGS-ko brains demonstrated also significant alterations in long-lasting effects evoked by pharmacological activation of glutamate receptors. Real-time RT-PCR revealed distinct patterns of dysregulated gene expression in the hippocampus and striatum of LGS-ko mice: LGS-ko hippocampus showed significantly modified expression of mRNAs for mGluR1, GluN2B subunit of NMDAR, and A1 adenosine receptors while altered expression of mRNAs for D1 dopamine receptors, the M1 cholinoreceptor and the acetylcholine-synthetizing enzyme choline-acetyltransferase was observed in LGS-ko striatum. Thus, inborn systemic hyperammonemia resulted in significant deficits in novelty acquisition and disturbed synaptic plasticity in corticostriatal and hippocampal pathways involved in learning and goal-directed behavior. PMID:28067279

  11. 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

  12. Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes.

    PubMed Central

    de Crécy-Lagard, V; Blanc, V; Gil, P; Naudin, L; Lorenzon, S; Famechon, A; Bamas-Jacques, N; Crouzet, J; Thibaut, D

    1997-01-01

    Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed. PMID:9006024

  13. Occurrence of Only One Form of Glutamine Synthetase in the Green Alga Monoraphidium braunii.

    PubMed Central

    Garcia-Fernandez, J. M.; Lopez-Ruiz, A.; Toribio, F.; Roldan, J. M.; Diez, J.

    1994-01-01

    Anion-exchange chromatography of crude extracts from the green alga Monoraphidium braunii yielded two glutamine synthetase (GS) activities. The ratio of activities was markedly different when crude extracts were subjected to various processing conditions but was not influenced by environmental factors of cell cultures. However, high performance liquid chromatography anion-exchange chromatograms showed only one GS if the crude extracts were processed immediately after cell disruption. Moreover, standard chromatography of crude extracts obtained in the absence of dithioerythritol, a reductant generally used in disruption buffers, yielded a single activity peak. Enzyme samples from the two activities obtained in the presence of dithioerythritol were purified for physicochemical characterization and antibody production. Both enzyme samples exhibited similar reactions to different inactivating agents and were undistinguishable by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Additionally, the two GS preparations showed absolute antigenic identity as demonstrated by immunodiffusion and immunoblotting experiments. Immunocytochemistry of M. braunii cryosections evidenced a chloroplast-specific distribution of the enzyme, which rules out the existence of a cytoplasmic counterpart. All these results support the proposal that M. braunii possesses only one form of GS. PMID:12232093

  14. Activities of nitrate reductase and glutamine synthetase in rice seedlings during cyanide metabolism.

    PubMed

    Yu, Xiao-Zhang; Zhang, Fu-Zhong

    2012-07-30

    A study was conducted to investigate activities of nitrate reductase (NR) and glutamine synthetase (GS) in plants during cyanide metabolism. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in the nutrient solutions containing KNO(3) or NH(4)Cl and treated with free cyanide (KCN). Cyanide in solutions and in plant materials was analyzed to estimate the phyto-assimilation potential. Activities of NR and GS in different parts of rice seedlings were assayed in vivo. Seedlings grown on NH(4)(+) showed significantly higher relative growth rate than those on NO(3)(-) (p<0.05) in the presence of exogenous cyanide. The metabolic rates of cyanide by seedlings were all positively correlated to the concentrations supplied. A negligible difference was observed between the two treatments with nitrate and ammonium (p>0.05). Enzymatic assays showed that cyanide (≥0.97mg CN L(-1)) impaired NR activity significantly in both roots and shoots (p<0.05). The effect of cyanide on GS activity in roots was more evident at 1.93mg CN L(-1), suggesting that NR activity was more susceptible to change from cyanide application than GS activity. The results observed here suggest that the exogenous cyanide, which to a certain level has a beneficial role in plant nutrition.

  15. Identification of a phosphinothricin-resistant mutant of rice glutamine synthetase using DNA shuffling

    PubMed Central

    Tian, Yong-Sheng; Xu, Jing; Zhao, Wei; Xing, Xiao-Juan; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2015-01-01

    To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resisitant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops. PMID:26492850

  16. Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster

    PubMed Central

    Cordonier, Elizabeth L.; Adjam, Riem; Camara Teixeira, Daniel; Onur, Simone; Zbasnik, Richard; Read, Paul E.; Döring, Frank; Schlegel, Vicki L.; Zempleni, Janos

    2015-01-01

    Holocarboxylase synthetase (HLCS) is the sole protein-biotin ligase in the human proteome. HLCS has key regulatory functions in intermediary metabolism, including fatty acid metabolism, and in gene repression through epigenetic mechanisms. The objective of this study was to identify foodborne inhibitors of HLCS that alter HLCS-dependent pathways in metabolism and gene regulation. When libraries of extracts from natural products and chemically pure compounds were screened for HLCS inhibitor activity, resveratrol compounds in grape materials caused an HLCS inhibition of >98% in vitro. The potency of these compounds was piceatannol > resveratrol > piceid. Grape-borne compounds other than resveratrol metabolites also contributed toward HLCS inhibition, e.g., p-coumaric acid and cyanidin chloride. HLCS inhibitors had meaningful effects on body fat mass. When Drosophila melanogaster brummer mutants, which are genetically predisposed to storing excess amounts of lipids, were fed diets enriched with grape leaf extracts and piceid, body fat mass decreased by more than 30% in males and females. However, Drosophila responded to inhibitor treatment with an increase in the expression of HLCS, which elicited an increase in the abundance of biotinylated carboxylases in vivo. We conclude that mechanisms other than inhibition of HLCS cause body fat loss in flies. We propose that the primary candidate is the inhibition of the insulin receptor/Akt signaling pathway. PMID:26303405

  17. Oxidative modification of glutamine synthetase by 2,2'-azobis(2- amidinopropane) dihydrochloride.

    PubMed

    Ma, Y S; Chao, C C; Stadtman, E R

    1999-03-01

    In the present study, we examined the pattern of protein modification elicited by alkylperoxyl radicals and alkylperoxides. To this end, we exposed glutamine synthetase (GS) and the peptide melittin to solutions containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which is known to decompose in aqueous, aerobic solutions to yield alkyl radicals and alkylperoxides. Under our conditions, pH 7.4, 37 degrees C, the AAPH-dependent formation of alkylhydroperoxide increased linearly with time and led to 40% inactivation of GS in 1 h and to complete inactivation in 4 h. Complete inactivation was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyrosine residues, 5 of 16 methionine residues, and all of the tryptophan residues (2 residues) per subunit. Inactivation of GS was associated also with some protein fragmentation and the formation of some higher molecular weight aggregates. Exposure of GS to AAPH led also to the generation of protein carbonyl derivatives (0.34 mol/mol subunit) and to formation of a significant amount (0.038 mol/mol subunits) of quinoprotein derivatives. To investigate the mechanism of tryptophan modification, the 26-amino-acid peptide, melittin, which contains one tryptophan but no histidine, tyrosine, or methionine residues, was treated with AAPH. N-Formylkynurenine was identified as the major product of tryptophan oxidation in melittin.

  18. A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast

    PubMed Central

    Mailu, Boniface M.; Ramasamay, Gowthaman; Mudeppa, Devaraja G.; Li, Ling; Lindner, Scott E.; Peterson, Megan J.; DeRocher, Amy E.; Kappe, Stefan H. I.; Rathod, Pradipsinh K.; Gardner, Malcolm J.

    2013-01-01

    The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNAGln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNAGlu and tRNAGln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNAGln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids. PMID:24072705

  19. Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product.

    PubMed

    Singh, Mangal; Chaudhary, Sandeep; Sareen, Dipti

    2017-03-01

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

  20. Glutamine synthetase in the phloem plays a major role in controlling proline production

    PubMed Central

    Brugiere, N; Dubois, F; Limami, AM; Lelandais, M; Roux, Y; Sangwan, RS; Hirel, B

    1999-01-01

    To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress. PMID:10521528

  1. 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.

  2. The role of glutamine synthetase in energy production and glutamine metabolism during oxidative stress.

    PubMed

    Aldarini, Nohaiah; Alhasawi, Azhar A; Thomas, Sean C; Appanna, Vasu D

    2017-01-17

    Oxidative stress is known to severely impede aerobic adenosine triphosphate (ATP) synthesis. However, the metabolically-versatile Pseudomonas fluorescens survives this challenge by invoking alternative ATP-generating networks. When grown in a medium with glutamine as the sole organic nutrient in the presence of H2O2, the microbe utilizes glutamine synthetase (GS) to modulate its energy budget. The activity of this enzyme that mediates the release of energy stored in glutamine was sharply increased in the stressed cells compared to the controls. The enhanced activities of such enzymes as acetate kinase, adenylate kinase and nucleotide diphosphate kinase ensured the efficacy of this ATP producing-machine by transferring the high energy phosphate. The elevated amounts of phosphoenol pyruvate carboxylase and pyruvate orthophosphate dikinase recorded in the H2O2 exposed cells provided another route to ATP independent of the reduction of O2. This is the first demonstration of a metabolic pathway involving GS dedicated to ATP synthesis. The phospho-transfer network that is pivotal to the survival of the microorganism under oxidative stress may reveal therapeutic targets against infectious microbes reliant on glutamine for their proliferation.

  3. Carnosine modulates glutamine synthetase expression in senescent astrocytes exposed to oxygen-glucose deprivation/recovery.

    PubMed

    Shi, Xiaojie; Wang, Bingyu; Liu, Yuan; Zhang, Jingjing; Huang, Yuyan; Cao, Pei; Shen, Yao; Lyu, Jianxin

    2017-01-20

    Carnosine is believed to be neuroprotective in cerebral ischemia. However, few reports concern its function on senescent astrocytes during cerebral ischemia. The aim of this study was to investigate the effects of carnosine on cell damage and glutamine synthetase (GS) expression in D-galactose-induced senescent astrocytes exposed to oxygen-glucose deprivation/recovery (OGD/R). The results showed that OGD/R caused massive cell damage and a significant decrease in GS expression both in the young and senescent astrocytes. The GS expression level was partly recovered whereas it continued to decline in the recovery stage in the young and senescent astrocytes, respectively. Decreased GS expression significantly inhibited glutamate uptake and glutamine production and release. Carnosine prevented the cell damage, rescued the expression of GS and reversed the glutamate uptake activity and glutamine production in the senescent astrocytes exposed to OGD/R. The modulatory effect of carnosine on GS expression was partly antagonized by pyrilamine, a selective histamine H1 receptors antagonist, but not bestatin. Bisindolylmaleimide II, a broad-spectrum inhibitor of PKC could also reverse the action of carnosine on GS expression. Thus, histamine H1 receptors and PKC pathway may be involved in the modulatory action of carnosine in GS expression in the senescent astrocytes exposed to OGD/R.

  4. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme.

    PubMed

    Seabra, Ana R; Carvalho, Helena G

    2015-01-01

    Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  5. Assessment of glutamine synthetase activity by [13N]ammonia uptake in living rat brain.

    PubMed

    Momosaki, Sotaro; Ito, Miwa; Tonomura, Misato; Abe, Kohji

    2015-01-01

    Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

  6. Influence of different host associations on glutamine synthetase activity and ammonium transporter in Santalumalbum L.

    PubMed

    Deepa, P; Yusuf, A

    2016-07-01

    The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica-sandal association compared to other leguminous and non-leguminous host associations. The association of N2 fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the [Formula: see text] method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.

  7. 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.

  8. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

  9. Secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis.

    PubMed

    Park, Min Chul; Kang, Taehee; Jin, Da; Han, Jung Min; Kim, Sang Bum; Park, Yun Jung; Cho, Kiwon; Park, Young Woo; Guo, Min; He, Weiwei; Yang, Xiang-Lei; Schimmel, Paul; Kim, Sunghoon

    2012-03-13

    Although adaptive systems of immunity against tumor initiation and destruction are well investigated, less understood is the role, if any, of endogenous factors that have conventional functions. Here we show that glycyl-tRNA synthetase (GRS), an essential component of the translation apparatus, circulates in serum and can be secreted from macrophages in response to Fas ligand that is released from tumor cells. Through cadherin (CDH)6 (K-cadherin), GRS bound to different ERK-activated tumor cells, and released phosphatase 2A (PP2A) from CDH6. The activated PP2A then suppressed ERK signaling through dephosphorylation of ERK and induced apoptosis. These activities were inhibited by blocking GRS with a soluble fragment of CDH6. With in vivo administration of GRS, growth of tumors with a high level of CDH6 and ERK activation were strongly suppressed. Our results implicate a conventional cytoplasmic enzyme in translation as an intrinsic component of the defense against ERK-activated tumor formation.

  10. Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene.

    PubMed Central

    Dixon, R

    1984-01-01

    Transcription of the structural gene for glutamine synthetase (glnA) in Klebsiella pneumoniae is controlled by the nitrogen regulatory genes ntrA, ntrB and ntrC. The nucleotide sequence of the regulatory region upstream of the glnA gene is reported here. High resolution S1 mapping of in vivo transcripts indicates that the regulatory region contains tandem promoters separated by 100 nucleotides. Measurements of beta-galactosidase activities determined in vivo from glnA-lac fusions suggest that the upstream promoter (for RNA2) is negatively regulated by the ntrBC gene products whereas transcription from the downstream promoter (for RNA1) is positively activated by the ntrA gene product in the presence of either the ntrBC or the nifA genes. The nucleotide sequence of the upstream promoter resembles the consensus sequence for E. coli promoters, whereas the downstream promoter shows homology with the nitrogen fixation (nif) promoters of K. pneumoniae. Images PMID:6149519

  11. Structural characterization of Helicobacter pylori dethiobiotin synthetase reveals differences between family members

    SciTech Connect

    Porebski, Przemyslaw J.; Klimecka, Maria; Chruszcz, Maksymilian; Nicholls, Robert A.; Murzyn, Krzysztof; Cuff, Marianne E.; Xu, Xiaohui; Cymborowski, Marcin; Murshudov, Garib N.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-07-11

    Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS-ATP and hpDTBS-GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.

  12. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

    PubMed

    Gudzera, Olga I; Golub, Andriy G; Bdzhola, Volodymyr G; Volynets, Galyna P; Lukashov, Sergiy S; Kovalenko, Oksana P; Kriklivyi, Ivan A; Yaremchuk, Anna D; Starosyla, Sergiy A; Yarmoluk, Sergiy M; Tukalo, Michail A

    2016-03-01

    Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6μM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27μM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01μM and IC90=13.53μM.

  13. Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

    SciTech Connect

    Elliott, J.I.; Ljungdahl, L.G.

    1982-04-01

    The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4'-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K/sub m/ values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4'-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20 to 30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20 to 30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

  14. Reculation of folylpolyglutamate synthetase in extracts of H35 hepatoma cells

    SciTech Connect

    Johnson, T.B.; Galivan, J.; Nair, M.G. )

    1987-05-01

    Folylpolyglutamate synthetase (FPGS) in extracts of H35 hepatoma cells was assayed using 250 {mu}M methotrexate (MTX) as the substrate under conditions where ({sup 3}H)glutamate incorporation was linear with respect to time and rotein concentration. Extracts from confluent cultures with reduced cellular folates exhibited nearly 1.7-fold higher FPGS specific activity than extracts of control cultures (600 pmol/hr/mg). Extracts of rapidly dividing cells (72 hrs) showed nearly a 2.3-fold increase. The addition of reduced exogenous folates such as folinic acid and 5-methyltetrahydrofolate (20 {mu}M, 24 hrs) to confluent cultures of folate-depleted cells typically lowered the FPGS activity in the resultant extracts by 40%, while a 42-hour exclusion of methionine from the media reduced the activity by half. The combination of methionine exclusion and folate addition for 42 hrs resulted in 75% lower FPGS activity vs extracts of control cultures of folate-depleted cells. These data suggest that the change sin the glutamylation rate of MTX in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. Using MTX or N{sup 10}-propargyl-5,8-dideazafolic acid (CB3717) as the starting substrate under appropriate assay conditions, FPGS from extracts catalyzed the formation of similar polyglutamate products as seen in analogous whole cell experiments.

  15. Large-scale filament formation inhibits the activity of CTP synthetase.

    PubMed

    Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

    2014-07-16

    CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

  16. In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

    PubMed Central

    Geslain, R; Martin, F; Delagoutte, B; Cavarelli, J; Gangloff, J; Eriani, G

    2000-01-01

    Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID:10744027

  17. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats.

    PubMed

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S; Simonsen, Mette; Ott, Peter; Vilstrup, Hendrik; Sørensen, Michael

    2014-03-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

  18. The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Sebastián, María; Arilla-Luna, Sonia; Baquedano, Silvia; Herguedas, Beatriz; Velázquez-Campoy, Adrián; Martínez-Júlvez, Marta; Medina, Milagros

    2017-03-24

    Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix α1c of the RFK module (positions K202, E203, F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix α1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites.

  19. Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

    PubMed Central

    2011-01-01

    Background There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in Drosophila. The function of SPS1, however, has not been elucidated. Results Differentially expressed genes in Drosophila SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after SPS1 knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by SPS1 knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by SPS1 knockdown and the formation of megamitochondria, the major phenotypic change observed by SPS1 knockdown. Conclusions These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities. PMID:21864351

  20. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  1. Glutamine synthetase stabilizes the binding of GlnR to nitrogen fixation gene operators.

    PubMed

    Fernandes, Gabriela de C; Hauf, Ksenia; Sant'Anna, Fernando H; Forchhammer, Karl; Passaglia, Luciane M P

    2017-03-01

    Biological nitrogen fixation (BNF) is a high energy demanding process carried out by diazotrophic microorganisms that supply combined nitrogen to the biosphere. The genes related to BNF are strictly regulated, but these mechanisms are poorly understood in gram-positive bacteria. The transcription factor GlnR was proposed to regulate nitrogen fixation-related genes based on Paenibacillus comparative genomics. In order to validate this proposal, we investigated BNF regulatory sequences in Paenibacillus riograndensis SBR5(T) genome. We identified GlnR-binding sites flanking σ(A) -binding sites upstream from BNF-related genes. GlnR binding to these sites was demonstrated by surface plasmon resonance spectroscopy. GlnR-DNA affinity is greatly enhanced when GlnR is in complex with feedback-inhibited (glutamine-occupied) glutamine synthetase (GS). GlnR-GS complex formation is also modulated by ATP and AMP. Thereby, gene repression exerted by the GlnR-GS complex is coupled with nitrogen (glutamine levels) and energetic status (ATP and AMP). Finally, we propose a DNA-looping model based on multiple operator sites that represents a strong and strict regulation for these genes.

  2. Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14.

    PubMed Central

    Shatters, R G; Somerville, J E; Kahn, M L

    1989-01-01

    Most rhizobia contain two glutamine synthetase (GS) enzymes: GSI, encoded by glnA, and GSII, encoded by glnII. We have found that WSU414, a Rhizobium meliloti 104A14 glutamine auxotroph derived from a glnA parental strain, is an ntrA mutant. The R. meliloti glnII promoter region contains DNA sequences similar to those found in front of other genes that require ntrA for their transcription. No GSII was found in the glnA ntrA mutant, and when a translational fusion of glnII to the Escherichia coli lacZ gene was introduced into WSU414, no beta-galactosidase was expressed. These results indicate that ntrA is required for glnII expression. The ntrA mutation did not prevent the expression of GSI. In free-living culture, the level of GSII and of the glnII-lacZ fusion protein was regulated by altering transcription in response to available nitrogen. No GSII protein was detected in alfalfa, pea, or soybean nodules when anti-GSII-specific antiserum was used. Images PMID:2570059

  3. 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

  4. Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases

    PubMed Central

    Tarry, Michael J.; Schmeing, T. Martin

    2015-01-01

    Nonribosomal peptide synthetases are large, multi-domain enzymes that produce peptide molecules with important biological activity such as antibiotic, antiviral, anti-tumor, siderophore and immunosuppressant action. The adenylation (A) domain catalyzes two reactions in the biosynthetic pathway. In the first reaction, it activates the substrate amino acid by adenylation and in the second reaction it transfers the amino acid onto the phosphopantetheine arm of the adjacent peptide carrier protein (PCP) domain. The conformation of the A domain differs significantly depending on which of these two reactions it is catalyzing. Recently, several structures of A–PCP di-domains have been solved using mechanism-based inhibitors to trap the PCP domain in the A domain active site. Here, we present an alternative strategy to stall the A–PCP di-domain, by engineering a disulfide bond between the native amino acid substrate and the A domain. Size exclusion studies showed a significant shift in apparent size when the mutant A–PCP was provided with cross-linking reagents, and this shift was reversible in the presence of high concentrations of reducing agent. The cross-linked protein crystallized readily in several of the conditions screened and the best crystals diffracted to ≈8 Å. PMID:25713404

  5. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  6. The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.

    PubMed

    Hewagama, A; Guy, H I; Chaparian, M; Evans, D R

    1998-11-10

    The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

  7. A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding.

    PubMed

    Zúñiga, Roberto; Salazar, Juan; Canales, Mauricio; Orellana, Omar

    2002-12-18

    The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.

  8. In Situ Glutamine Synthetase Activity in a Marine Unicellular Alga (Development of a Sensitive Colorimetric Assay and the Effects of Nitrogen Status on Enzyme Activity).

    PubMed Central

    Rees, TAV.; Larson, T. R.; Heldens, JWG.; Huning, FGJ.

    1995-01-01

    A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N[prime]-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 [mu]M, but this value decreased to 110 [mu]M with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20[deg]C). PMID:12228676

  9. Probing the global and local dynamics of aminoacyl-tRNA synthetases using all-atom and coarse-grained simulations.

    PubMed

    Strom, Alexander M; Fehling, Samuel C; Bhattacharyya, Sudeep; Hati, Sanchita

    2014-05-01

    Coarse-grained simulations have emerged as invaluable tools for studying conformational changes in biomolecules. To evaluate the effectiveness of computationally inexpensive coarse-grained models in studying global and local dynamics of large protein systems like aminoacyl-tRNA synthetases, we have performed coarse-grained normal mode analysis, as well as principle component analysis on trajectories of all-atom and coarse-grained molecular dynamics simulations for three aminoacyl-tRNA synthetases--Escherichia coli methionyl-tRNA synthetase, Thermus thermophilus leucyl-tRNA synthetase, and Enterococcus faecium prolyl-tRNA synthetase. In the present study, comparison of predicted dynamics based on B-factor and overlap calculations revealed that coarse-grained methods are comparable to the all-atom simulations in depicting the intrinsic global dynamics of the three enzymes. However, the principal component analyses of the motions obtained from the all-atom molecular dynamics simulations provide a superior description of the local fluctuations of these enzymes. In particular, the all-atom model was able to capture the functionally relevant substrate-induced dynamical changes in prolyl-tRNA synthetase. The alteration in the coupled dynamics between the catalytically important proline-binding loop and its neighboring structural elements due to substrate binding has been characterized and reported for the first time. Taken together, the study portrays comparable and contrasting situations in studying the functional dynamics of large multi-domain aminoacyl-tRNA synthetases using coarse-grained and all-atom simulation methods.

  10. AC Losses of Prototype HTS Transmission Cables

    SciTech Connect

    Demko, J.A.; Dresner, L.; Hughey, R.L.; Lue, J.W.; Olsen, S.K.; Sinha, U.; Tolbert, J.C.

    1998-09-13

    Since 1995 Southwire Company and Oak Ridge National Laboratory (ORNL) have jointly designed, built, and tested nine, l-m long, high temperature superconducting (HTS) transmission cable prototypes. This paper summarizes the AC loss measurements of five of the cables not reported elsewhere, and compares the losses with each other and with theory developed by Dresner. Losses were measured with both a calorimetric and an electrical technique. Because of the broad resistive transition of the HTS tapes, the cables can be operated stably beyond their critical currents. The AC losses were measured in this region as well as below critical currents. Dresner's theory takes into account the broad resistive transition of the HTS tapes and calculates the AC losses both below and above the critical current. The two sets of AC 10SS data agree with each other and with the theory quite welL In particular, at low currents of incomplete penetration, the loss data agree with the theoretical prediction of hysteresis loss based on only the outer two Iayers carrying the total current.

  11. AC power generation from microbial fuel cells

    NASA Astrophysics Data System (ADS)

    Lobo, Fernanda Leite; Wang, Heming; Forrestal, Casey; Ren, Zhiyong Jason

    2015-11-01

    Microbial fuel cells (MFCs) directly convert biodegradable substrates to electricity and carry good potential for energy-positive wastewater treatment. However, the low and direct current (DC) output from MFC is not usable for general electronics except small sensors, yet commercial DC-AC converters or inverters used in solar systems cannot be directly applied to MFCs. This study presents a new DC-AC converter system for MFCs that can generate alternating voltage in any desired frequency. Results show that AC power can be easily achieved in three different frequencies tested (1, 10, 60 Hz), and no energy storage layer such as capacitors was needed. The DC-AC converter efficiency was higher than 95% when powered by either individual MFCs or simple MFC stacks. Total harmonic distortion (THD) was used to investigate the quality of the energy, and it showed that the energy could be directly usable for linear electronic loads. This study shows that through electrical conversion MFCs can be potentially used in household electronics for decentralized off-grid communities.

  12. Organic magnetoresistance under resonant ac drive

    NASA Astrophysics Data System (ADS)

    Roundy, R. C.; Raikh, M. E.

    2013-09-01

    Motivated by a recent experiment, we develop a theory of organic magnetoresistance (OMAR) in the presence of a resonant ac drive. To this end, we perform a thorough analysis of the dynamics of ac-driven electron-hole polaron pair in magnetic field, which is a sum of external and random hyperfine fields. Resonant ac drive affects the OMAR by modifying the singlet content of the eigenmodes. This, in turn, leads to the change of recombination rate, and ultimately, to the change of the spin-blocking that controls the current. Our analysis demonstrates that, upon increasing the drive amplitude, the blocking eigenmodes of the triplet type acquire a singlet admixture and become unblocking. Most surprisingly, the opposite process goes in parallel: new blocking modes emerge from nonblocking precursors as the drive increases. These emergent blocking modes are similar to subradiant modes in the Dicke effect. A nontrivial evolution of eigenmodes translates into a nontrivial behavior of OMAR with the amplitude of the ac drive: it is initially linear, then passes through a maximum, drops, and finally saturates.

  13. A dry-cooled AC quantum voltmeter

    NASA Astrophysics Data System (ADS)

    Schubert, M.; Starkloff, M.; Peiselt, K.; Anders, S.; Knipper, R.; Lee, J.; Behr, R.; Palafox, L.; Böck, A. C.; Schaidhammer, L.; Fleischmann, P. M.; Meyer, H.-G.

    2016-10-01

    The paper describes a dry-cooled AC quantum voltmeter system operated up to kilohertz frequencies and 7 V rms. A 10 V programmable Josephson voltage standard (PJVS) array was installed on a pulse tube cooler (PTC) driven with a 4 kW air-cooled compressor. The operating margins at 70 GHz frequencies were investigated in detail and found to exceed 1 mA Shapiro step width. A key factor for the successful chip operation was the low on-chip power consumption of 65 mW in total. A thermal interface between PJVS chip and PTC cold stage was used to avoid a significant chip overheating. By installing the cryocooled PJVS array into an AC quantum voltmeter setup, several calibration measurements of dc standards and calibrator ac voltages up to 2 kHz frequencies were carried out to demonstrate the full functionality. The results are discussed and compared to systems with standard liquid helium cooling. For dc voltages, a direct comparison measurement between the dry-cooled AC quantum voltmeter and a liquid-helium based 10 V PJVS shows an agreement better than 1 part in 1010.

  14. 48 CFR Appendixes A-C to Chapter 7 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false A Appendixes A-C to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes A-C to Chapter 7...

  15. 48 CFR Appendixes A-C to Chapter 7 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false A Appendixes A-C to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes A-C to Chapter 7...

  16. AC losses in a HTS coil carrying DC current in AC external magnetic field

    NASA Astrophysics Data System (ADS)

    Ogawa, J.; Zushi, Y.; Fukushima, M.; Tsukamoto, O.; Suzuki, E.; Hirakawa, M.; Kikukawa, K.

    2003-10-01

    We electrically measured AC losses in a Bi2223/Ag-sheathed pancake coil excited by a DC current in AC external magnetic field. Losses in the coil contain two kinds of loss components that are the magnetization losses and dynamic resistance losses. In the measurement, current leads to supply a current to the coil were specially arranged to suppress electromagnetic coupling between the coil current and the AC external magnetic field. A double pick-up coils method was used to suppress a large inductive voltage component contained in voltage signal for measuring the magnetization losses. It was observed that the magnetization losses were dependent on the coil current and that a peak of a curve of the loss factor vs. amplitude of the AC external magnetic field shifted to lower amplitude of the AC magnetic field as the coil current increased. This result suggests the full penetration magnetic field of the coil tape decreases as the coil current increases. The dynamic resistance losses were measured by measuring a DC voltage appearing between the coil terminals. It was observed that the DC voltage appearing in the coil subject to the AC external magnetic field was much larger than that in the coil subject to DC magnetic field.

  17. Evaluation of modern IGBT-modules for hard-switched AC/DC/AC converters

    SciTech Connect

    Blaabjerg, F.; Pedersen, J.K.; Jaeger, U.

    1995-12-31

    The development of IGBT devices is still producing faster devices with lower losses. The applications become more advanced like a complete hard-switched AC/DC/AC converter with almost clean input current and regenerating capabilities. This paper will first focus on a detailed characterization and comparison of eight different IGBT-modules representing state-of-the-art for both PT and NPT technologies. The voltage level of the devices is 1,200V and 1,600V/1,700V. The characterization is done on an advanced measurement system which is briefly described. The characterization is based on static and dynamic tests for both IGBT and the diodes in the IGBT-modules at a junction temperature at 125 C. The comparison is first done directly based on conduction losses and switching losses, and later the measurements are used in a loss model for a complete AC/DC/AC converter application. In the AC/DC/AC converter the power losses are modelled, and different operating conditions are compared like different voltage levels in the DC-link. It is concluded dependent on operation conditions different devices will be preferable, but the high voltage devices have the highest losses even at a high operating voltage.

  18. 7 CFR 1737.31 - Area Coverage Survey (ACS).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 11 2012-01-01 2012-01-01 false Area Coverage Survey (ACS). 1737.31 Section 1737.31... Studies-Area Coverage Survey and Loan Design § 1737.31 Area Coverage Survey (ACS). (a) The Area Coverage Survey (ACS) is a market forecast of service requirements of subscribers in a proposed service area....

  19. 7 CFR 1737.31 - Area Coverage Survey (ACS).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Area Coverage Survey (ACS). 1737.31 Section 1737.31... Studies-Area Coverage Survey and Loan Design § 1737.31 Area Coverage Survey (ACS). (a) The Area Coverage Survey (ACS) is a market forecast of service requirements of subscribers in a proposed service area....

  20. 7 CFR 1737.31 - Area Coverage Survey (ACS).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 11 2013-01-01 2013-01-01 false Area Coverage Survey (ACS). 1737.31 Section 1737.31... Studies-Area Coverage Survey and Loan Design § 1737.31 Area Coverage Survey (ACS). (a) The Area Coverage Survey (ACS) is a market forecast of service requirements of subscribers in a proposed service area....

  1. 7 CFR 1737.31 - Area Coverage Survey (ACS).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 11 2011-01-01 2011-01-01 false Area Coverage Survey (ACS). 1737.31 Section 1737.31... Studies-Area Coverage Survey and Loan Design § 1737.31 Area Coverage Survey (ACS). (a) The Area Coverage Survey (ACS) is a market forecast of service requirements of subscribers in a proposed service area....

  2. 21 CFR 880.5500 - AC-powered patient lift.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false AC-powered patient lift. 880.5500 Section 880.5500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Devices § 880.5500 AC-powered patient lift. (a) Identification. An AC-powered lift is an...

  3. Methods for Addressing Missing Data with Applications from ACS Exams

    ERIC Educational Resources Information Center

    Brandriet, Alexandra; Holme, Thomas

    2015-01-01

    As part of the ACS Examinations Institute (ACS-EI) national norming process, student performance data sets are collected from professors at colleges and universities from around the United States. Because the data sets are collected on a volunteer basis, the ACS-EI often receives data sets with only students' total scores and without the students'…

  4. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  5. Arabidopsis Plastidial Folylpolyglutamate Synthetase Is Required for Seed Reserve Accumulation and Seedling Establishment in Darkness

    PubMed Central

    Meng, Hongyan; Jiang, Ling; Xu, Bosi; Guo, Wenzhu; Li, Jinglai; Zhu, Xiuqing; Qi, Xiaoquan; Duan, Lixin; Meng, Xianbin; Fan, Yunliu; Zhang, Chunyi

    2014-01-01

    Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3−. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3− conditions, and further enhanced under NO3− limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3− during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3− as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis. PMID:25000295

  6. Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.

    PubMed

    Wu, C

    1977-04-01

    Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and alpha-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the gamma-glutamyl transfer reaction of the enzyme. Inhibition of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.

  7. Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

    PubMed

    Mudge, S J; Williams, J H; Eyre, H J; Sutherland, G R; Cowan, P J; Power, D A

    1998-03-16

    Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

  8. Metabolic indicators of drought stress tolerance in wheat: glutamine synthetase isoenzymes and Rubisco.

    PubMed

    Nagy, Zoltán; Németh, Edit; Guóth, Adrienn; Bona, Lajos; Wodala, Barnabás; Pécsváradi, Attila

    2013-06-01

    Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance.

  9. Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots.

    PubMed

    Konishi, Noriyuki; Ishiyama, Keiki; Beier, Marcel Pascal; Inoue, Eri; Kanno, Keiichi; Yamaya, Tomoyuki; Takahashi, Hideki; Kojima, Soichi

    2016-12-21

    Glutamine synthetase (GS) catalyzes a reaction that incorporates ammonium into glutamate and yields glutamine in the cytosol and chloroplasts. Although the enzymatic characteristics of the GS1 isozymes are well known, their physiological functions in ammonium assimilation and regulation in roots remain unclear. In this study we show evidence that two cytosolic GS1 isozymes (GLN1;2 and GLN1;3) contribute to ammonium assimilation in Arabidopsis roots. Arabidopsis T-DNA insertion lines for GLN1;2 and GLN1;3 (i.e. gln1;2 and gln1;3 single-mutants), the gln1;2:gln1;3 double-mutant, and the wild-type accession (Col-0) were grown in hydroponic culture with variable concentrations of ammonium to compare their growth, and their content of nitrogen, carbon, ammonium, and amino acids. GLN1;2 and GLN1;3 promoter-dependent green fluorescent protein was observed under conditions with or without ammonium supply. Loss of GLN1;2 caused significant suppression of plant growth and glutamine biosynthesis under ammonium-replete conditions. In contrast, loss of GLN1;3 caused slight defects in growth and Gln biosynthesis that were only visible based on a comparison of the gln1;2 single- and gln1;2:gln1;3 double-mutants. GLN1;2, being the most abundantly expressed GS1 isozyme, markedly increased following ammonium supply and its promoter activity was localized at the cortex and epidermis, while GLN1;3 showed only low expression at the pericycle, suggesting their different physiological contributions to ammonium assimilation in roots. The GLN1;2 promoter-deletion analysis identified regulatory sequences required for controlling ammonium-responsive gene expression of GLN1;2 in Arabidopsis roots. These results shed light on GLN1 isozyme-specific regulatory mechanisms in Arabidopsis that allow adaptation to an ammonium-replete environment.

  10. Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes.

    PubMed

    Laberge, Tammy; Walsh, Patrick J

    2011-06-01

    Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate from glutamine or ammonia, bicarbonate and ATP. There are three different isoforms of CPS that play vital roles in two metabolic pathways, pyrimidine biosynthesis (CPS II) and arginine/urea biosynthesis (CPS I and CPS III). Gene duplication has been proposed as the evolutionary mechanism creating this gene family with CPS II likely giving rise to the CPS I/III clade. In the evolutionary history of this gene family it is still undetermined when CPS I diverged from CPS III on the path to terrestriality in the vertebrates. Transitional organisms such as lungfishes are of particular interest because they are capable of respiring via gills and with lungs and therefore can be found in both aquatic and terrestrial environments. Notably, enzymatic characterization of the mitochondrial CPS isoforms in this transitional group has not led to clear conclusions. In order to determine which CPS isoform is present in transitional animals, we examined partial sequences for liver CPS amplified from five species of lungfish, and a larger fragment of CPS from one lungfish species (Protopterus annectens) and compared them to CPS isoforms from other fish and mammals. Enzyme activities for P. annectens liver were also examined. While enzyme activities did not yield a clear distinction between isoforms (virtually equal activities were obtained for either CPS I or III), CPS sequences from the lungfishes formed a monophyletic clade within the CPS I clade and separate from the CPS III clade of other vertebrates. This finding implies that the mitochondrial isoform of CPS in lungfish is derived from CPS I and is likely to have a physiological function similar to CPS I. This finding is important because it supports the hypothesis that lungfish employ a urea cycle similar to terrestrial air-breathing vertebrates.

  11. Function of a glutamine synthetase-like protein in bacterial aniline oxidation via γ-glutamylanilide.

    PubMed

    Takeo, Masahiro; Ohara, Akira; Sakae, Shinji; Okamoto, Yasuhiro; Kitamura, Chitoshi; Kato, Dai-ichiro; Negoro, Seiji

    2013-10-01

    Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.

  12. Astrocyte glutamine synthetase: importance in hyperammonemic syndromes and potential target for therapy.

    PubMed

    Brusilow, Saul W; Koehler, Raymond C; Traystman, Richard J; Cooper, Arthur J L

    2010-10-01

    Many theories have been advanced to explain the encephalopathy associated with chronic liver disease and with the less common acute form. A major factor contributing to hepatic encephalopathy is hyperammonemia resulting from portacaval shunting and/or liver damage. However, an increasing number of causes of hyperammonemic encephalopathy have been discovered that present with the same clinical and laboratory features found in acute liver failure, but without liver failure. Here, we critically review the physiology, pathology, and biochemistry of ammonia (i.e., NH3 plus NH4+) and show how these elements interact to constitute a syndrome that clinicians refer to as hyperammonemic encephalopathy (i.e., acute liver failure, fulminant hepatic failure, chronic liver disease). Included will be a brief history of the status of ammonia and the centrality of the astrocyte in brain nitrogen metabolism. Ammonia is normally detoxified in the liver and extrahepatic tissues by conversion to urea and glutamine, respectively. In the brain, glutamine synthesis is largely confined to astrocytes, and it is generally accepted that in hyperammonemia excess glutamine compromises astrocyte morphology and function. Mechanisms postulated to account for this toxicity will be examined with emphasis on the osmotic effects of excess glutamine (the osmotic gliopathy theory). Because hyperammonemia causes osmotic stress and encephalopathy in patients with normal or abnormal liver function alike, the term "hyperammonemic encephalopathy" can be broadly applied to encephalopathy resulting from liver disease and from various other diseases that produce hyperammonemia. Finally, the possibility that a brain glutamine synthetase inhibitor may be of therapeutic benefit, especially in the acute form of liver disease, is discussed.

  13. Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

    PubMed Central

    Bender, Robert A.; Streicher, Stanley L.

    1979-01-01

    We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5′-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images PMID:33958

  14. Identification and expression analyses of cytosolic glutamine synthetase genes in barley (Hordeum vulgare L.).

    PubMed

    Goodall, Andrew J; Kumar, Pankaj; Tobin, Alyson K

    2013-04-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, particularly during seed development. Three cytosolic GS isoforms (HvGS1) were identified in barley (Hordeum vulgare L. cv Golden Promise). Quantitation of gene expression, localization and response to N supply revealed that each gene plays a non-redundant role in different tissues and during development. Localization of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that it is important in N transport and remobilization. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localized to the leaf mesophyll cells, in the cortex and pericycle of roots, and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in leaves with an increasing supply of N, suggesting its role in the primary assimilation of N. HvGS1_3 was specifically and predominantly localized in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH(+)4, suggesting that it has a primary role in grain N assimilation and also in the protection against ammonium toxicity in roots. The expression of HvGS1 genes is directly correlated with protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley.

  15. 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.

  16. Nonribosomal biosynthesis of vancomycin-type antibiotics: a heptapeptide backbone and eight peptide synthetase modules.

    PubMed

    Recktenwald, Jürgen; Shawky, Riham; Puk, Oliver; Pfennig, Frank; Keller, Ulrich; Wohlleben, Wolfgang; Pelzer, Stefan

    2002-04-01

    During analysis of the recently identified gene cluster for the glycopeptide antibiotic balhimycin, produced by Amycolatopsis mediterranei DSM 5908, novel genes were identified and characterized in detail. The gene products of four of the identified genes (bpsA, bpsB, bpsC and bpsD) are nonribosomal peptide synthetases (NRPSs); one (Orf1-protein) shows similarities to small proteins associated with several NRPSs without an assigned function. BpsA and BpsB are composed of three modules each (modules 1-6), BpsC of one module (module 7) and BpsD of a minimal module (module 8). Thus, the balhimycin gene cluster encodes eight modules, whereas its biosynthetic product is a heptapeptide. Non-producing mutants were created by a gene disruption of bpsB, an in-frame deletion of bpsC and a gene replacement of bpsD. After establishment of a gene complementation system for Amycolatopsis strains, the replacement mutant of bpsD was complemented, demonstrating for the first time that BpsD, encoding the eighth module, is indeed involved in balhimycin biosynthesis. After feeding with beta-hydroxytyrosine the capability of the bpsD mutant to produce balhimycin was restored, demonstrating the participation of BpsD in the biosynthesis of this amino acid. The specificity of four of the eight adenylation domains was determined by ATP/PP(i) exchange assays: modules 4 and 5 activated L-4-hydroxyphenylglycine, module 6 activated beta-hydroxytyrosine and module 7 activated L-3,5-dihydroxyphenylglycine, which is in accordance with the sequence of the non-proteogenic amino acids 4 to 7 of the balhimycin backbone.

  17. CMP-N-acetylneuraminic acid synthetase interacts with fragile X related protein 1

    PubMed Central

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-01-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. PMID:27357083

  18. Methionyl-tRNA synthetase from Caenorhabditis elegans: A specific multidomain organization for convergent functional evolution

    PubMed Central

    Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

    2010-01-01

    Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl-tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA–protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA–protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes. PMID:20954242

  19. Dual targeting of aminoacyl-tRNA synthetases to the mitochondrion and complex plastid in chlorarachniophytes.

    PubMed

    Hirakawa, Yoshihisa; Burki, Fabien; Keeling, Patrick J

    2012-12-15

    In plants, many nucleus-encoded proteins are targeted to both mitochondria and plastids, and this process is generally mediated by ambiguous N-terminal targeting sequences that are recognized by receptors on both organelles. In many algae, however, plastids were acquired by secondarily engulfing green or red algae, which were retained within the endomembrane system. Protein targeting to these secondary plastids is more complex, and because they do not reside directly in the cytoplasm, dual targeting cannot function as it does in plant cells. Here we investigate dual targeting of aminoacyl-tRNA synthetases (aaRSs) in chlorarachniophytes, which are complex algae that possess secondary plastids and a relict nucleus derived from a green algal endosymbiont. Chlorarachniophytes have four genome-containing compartments, but almost all the aaRSs are nucleus-encoded and present in fewer than four copies (some as few as two), suggesting multiple targeting. We characterized the subcellular localization of two classes, HisRS (three copies) and GlyRS (two copies), using GFP fusion proteins. In both cases, one copy was dually targeted to mitochondria and plastids, but unlike plants this was mediated by translation initiation variants. We also found that the periplastidal compartment (the relict green algal cytoplasm) lacks both GlyRS and a cognate tRNA, suggesting that pre-charged host tRNAs are imported into this compartment. Leader analysis of other aaRSs suggests that alternative translation is a common strategy for dual targeting in these complex cells. Overall, dual targeting to mitochondria and plastids is a shared feature of plastid-bearing organisms, but the increased complexity of trafficking into secondary plastids requires a different strategy.

  20. 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