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Sample records for acyl-coa synthetase activities

  1. Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

    PubMed

    Nchoutmboube, Jules A; Viktorova, Ekaterina G; Scott, Alison J; Ford, Lauren A; Pei, Zhengtong; Watkins, Paul A; Ernst, Robert K; Belov, George A

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  2. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  3. Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

    PubMed

    Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

    2009-08-01

    Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

  4. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids.

    PubMed

    Oakes, Ken D; Sibley, Paul K; Martin, Jon W; MacLean, Dan D; Solomon, Keith R; Mabury, Scott A; Van Der Kraak, Glen J

    2005-05-01

    This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

  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. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  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. [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)

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  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. 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. 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

  12. 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

  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. 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. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Succinyl-CoA synthetase is a phosphate target for the activation of mitochondrial metabolism.

    PubMed

    Phillips, Darci; Aponte, Angel M; French, Stephanie A; Chess, David J; Balaban, Robert S

    2009-08-04

    Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism, and heme synthesis. Inorganic phosphate (P(i)) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study, it was shown that P(i) binds the porcine heart SCS alpha-subunit (SCSalpha) in a noncovalent manner and enhances its enzymatic activity, thereby providing a new target for P(i) activation in mitochondria. Coupling 32P labeling of intact mitochondria with SDS gel electrophoresis revealed that 32P labeling of SCSalpha was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS), we showed that this enhanced 32P labeling resulted from a simple binding of 32P, not covalent protein phosphorylation. The ability of SCSalpha to retain its 32P throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a P(i)-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with millimolar concentrations of P(i). Since the level of 32P binding to SCSalpha was increased in substrate-depleted mitochondria, where the matrix P(i) concentration is increased, we conclude that SCS activation by P(i) binding represents another mitochondrial target for the P(i)-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria.

  4. 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.

  5. 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

  6. 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

  7. Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The purification and properties of an endogenous activator of the enzyme.

    PubMed

    Neuberger, A; Sandy, J D; Tait, G H

    1973-11-01

    1. A low-molecular-weight activator of 5-aminolaevulinate synthetase was detected in extracts of Rhodopseudomonas spheroides. The compound activates the enzyme extracted from oxygenated semi-anaerobically grown organisms by a factor of 6-8. 2. The activator was extensively purified, but owing to the exceedingly small amounts that could be extracted in the active form its structure was not determined. 3. The activator contains an acetylatable amino group; it is more stable at acid than at alkaline pH values; it is stable to treatment with I(2)-KI or potassium ferricyanide, but irreversibly inactivated by Na(2)S(2)O(4) or NaBH(4). 4. The chromatographic, electrophoretic, chemical and stability properties of the activator are similar to those of pteridines; purified activator preparations contain pteridines, as shown by their fluorescence spectrum. This does not, however, constitute an identification of the activator. 5. The activator enhances the activity of crude and partially purified enzyme and does not appear to require other endogenous factors or a supply of air to produce activation. Activation of the purified enzyme, however, requires the presence of either pyridoxal phosphate or sodium succinate. In the absence of both these factors the activator produces a time- and temperature-dependent decay of activity.

  8. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  9. Progressive neuronal activation accompanies epileptogenesis caused by hippocampal glutamine synthetase inhibition.

    PubMed

    Albright, Benjamin; Dhaher, Roni; Wang, Helen; Harb, Roa; Lee, Tih-Shih W; Zaveri, Hitten; Eid, Tore

    2017-02-01

    Loss of glutamine synthetase (GS) in hippocampal astrocytes has been implicated in the causation of human mesial temporal lobe epilepsy (MTLE). However, the mechanism by which the deficiency in GS leads to epilepsy is incompletely understood. Here we ask how hippocampal GS inhibition affects seizure phenotype and neuronal activation during epilepsy development (epileptogenesis). Epileptogenesis was induced by infusing the irreversible GS blocker methionine sulfoximine (MSO) unilaterally into the hippocampal formation of rats. We then used continuous video-intracranial electroencephalogram (EEG) monitoring and c-Fos immunohistochemistry to determine the type of seizures and spatial distribution of neuronal activation early (1-5days postinfusion) and late (16-43days postinfusion) in epileptogenesis. Early in epileptogenesis, seizures were preferentially mild (stage 1-2), activating neurons in the entorhinal-hippocampal area, the basolateral amygdala, the piriform cortex, the midline thalamus, and the anterior olfactory area. Late in epileptogenesis, the seizures were generally more severe (stages 4-5) with neuronal activation extending to the neocortex, the bed nucleus of the stria terminalis, the mediodorsal thalamu\\s, and the central nucleus of the amygdala. Our findings demonstrate that inhibition of GS focally in the hippocampal formation triggers a process of epileptogenesis characterized by gradual worsening of seizure severity and involvement of progressively larger neuronal populations over a period of several weeks. Knowledge about the underlying mechanism of epileptogenesis is important because such knowledge may result in more specific and efficacious treatments of MTLE by moving away from large and poorly specific surgical resections to highly targeted surgical or pharmacological interventions of the epileptogenic process.

  10. 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.

  11. Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

    PubMed Central

    Stoughton, D M; Zapata, G; Picone, R; Vann, W F

    1999-01-01

    Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding. PMID:10510306

  12. 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.

  13. 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.

  14. Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase.

    PubMed

    Liu, Ru-Juan; Tan, Min; Du, Dao-Hai; Xu, Bei-Si; Eriani, Gilbert; Wang, En-Duo

    2011-12-01

    A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

  15. 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

  16. Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin.

    PubMed

    Demant, E J; Nystrøm, B T

    2001-08-01

    The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.

  17. Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells.

    PubMed

    Weedon-Fekjaer, M Susanne; Dalen, Knut Tomas; Solaas, Karianne; Staff, Anne Cathrine; Duttaroy, Asim K; Nebb, Hilde Irene

    2010-07-01

    Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

  18. 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

  19. Quantitative structure-activity relationship studies of a series of sulfa drugs as inhibitors of Pneumocystis carinii dihydropteroate synthetase.

    PubMed

    Johnson, T; Khan, I A; Avery, M A; Grant, J; Meshnick, S R

    1998-06-01

    Sulfone and sulfanilamide sulfa drugs have been shown to inhibit dihydropteroate synthetase (DHPS) isolated from Pneumocystis carinii. In order to develop a pharmacophoric model for this inhibition, quantitative structure-activity relationships (QSAR) for sulfa drugs active against DHPS have been studied. Accurate 50% inhibitory concentrations were collected for 44 analogs, and other parameters, such as partition coefficients and molar refractivity, were calculated. Conventional multiple regression analysis of these data did not provide acceptable QSAR. However, three-dimensional QSAR provided by comparative molecular field analysis did give excellent results. Upon removal of poorly correlated analogs, a data set of 36 analogs, all having a common NHSO2 group, provided a cross-validated r2 value of 0.699 and conventional r2 value of 0.964. The resulting pharmacophore model should be useful for understanding and predicting the binding of DHPS by new sulfa drugs.

  20. Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus.

    PubMed

    Chungopast, Sirinapa; Thapanapongworakul, Pilunthana; Matsuura, Hiroyuki; Van Dao, Tan; Asahi, Toshimasa; Tada, Kuninao; Tajima, Shigeyuki; Nomura, Mika

    2014-03-01

    In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume-rhizobia symbiosis by modifying nodule development and metabolic processes.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-05-01

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

  3. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-12-11

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

  4. Total Glutamine Synthetase Activity during Soybean Nodule Development Is Controlled at the Level of Transcription and Holoprotein Turnover.

    PubMed Central

    Temple, S. J.; Kunjibettu, S.; Roche, D.; Sengupta-Gopalan, C.

    1996-01-01

    Gln synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to yield Gln. In higher plants GS is an octameric enzyme and the subunits are encoded by members of a small multigene family. In soybeans (Glycine max), following the onset of N2 fixation there is a dramatic increase in GS activity in the root nodules. GS activity staining of native polyacrylamide gels containing nodule and root extracts showed a common band of activity (GSrs). The nodules also contained a slower-migrating, broad band of enzyme activity (GSns). The GSns activity band is a complex of many isozymes made up of different proportions of two kinds of GS subunits: GSr and GSn. Root nodules formed following inoculation with an Nif- strain of Bradyrhizobium japonicum showed the presence of GS isoenzymes (GSns1) with low enzyme activity, which migrated more slowly than GSns. Gsns1 is most likely made up predominantly of GSn subunits. Our data suggest that, whereas the class I GS genes encoding the GSr subunits are regulated by the availability of NH3, the class II GS genes coding for the GSn subunits are developmentally regulated. Furthermore, we have demonstrated that the GSns1 isozymes in the Nif- nodules are relatively more labile. Our overall conclusion is that GSns activity in soybean nodules is regulated by N2 fixation both at the level of transcription and at the level of holoprotein stability. PMID:12226474

  5. Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

    SciTech Connect

    Knight, T.J.; Temple, S.; Sengupta-Gopalan, C.

    1996-05-15

    Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels of GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.

  6. 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

  7. 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

  8. Modulating effects of acyl-CoA synthetase 5-derived mitochondrial Wnt2B palmitoylation on intestinal Wnt activity

    PubMed Central

    Klaus, Christina; Schneider, Ursula; Hedberg, Christian; Schütz, Anke K; Bernhagen, Jürgen; Waldmann, Herbert; Gassler, Nikolaus; Kaemmerer, Elke

    2014-01-01

    AIM: To investigate the role of acyl-CoA synthetase 5 (ACSL5) activity in Wnt signaling in intestinal surface epithelia. METHODS: Several cell lines were used to investigate the ACSL5-dependent expression and synthesis of Wnt2B, a mitochondrially expressed protein of the Wnt signaling family. Wnt activity was functionally assessed with a luciferase reporter assay. ACSL5-related biochemical Wnt2B modifications were investigated with a modified acyl-exchange assay. The findings from the cell culture models were verified using an Apcmin/+ mouse model as well as normal and neoplastic diseased human intestinal tissues. RESULTS: In the presence of ACSL5, Wnt2B was unable to translocate into the nucleus and was enriched in mitochondria, which was paralleled by a significant decrease in Wnt activity. ACSL5-dependent S-palmitoylation of Wnt2B was identified as a molecular reason for mitochondrial Wnt2B accumulation. In cell culture systems, a strong relation of ACSL5 expression, Wnt2B palmitoylation, and degree of malignancy were found. Using normal mucosa, the association of ACSL5 and Wnt2B was seen, but in intestinal neoplasias the mechanism was only rudimentarily observed. CONCLUSION: ACSL5 mediates antiproliferative activities via Wnt2B palmitoylation with diminished Wnt activity. The molecular pathway is probably relevant for intestinal homeostasis, overwhelmed by other pathways in carcinogenesis. PMID:25356045

  9. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

    PubMed Central

    Recuero-Checa, Maria A.; Sharma, Manu; Lau, Constance; Watkins, Paul A.; Gaydos, Charlotte A.; Dean, Deborah

    2016-01-01

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs. PMID:26988341

  10. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs).

    PubMed

    Recuero-Checa, Maria A; Sharma, Manu; Lau, Constance; Watkins, Paul A; Gaydos, Charlotte A; Dean, Deborah

    2016-03-18

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

  11. Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism

    PubMed Central

    Golovko, Mikhail Y.; Rosenberger, Thad A.; Faergeman, Nils J.; Feddersen, Søren; Cole, Nelson B.; Pribill, Ingrid; Berger, Johannes; Nussbaum, Robert L.; Murphy, Eric J.

    2008-01-01

    Because α-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of α-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-14C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetases (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wt mouse or human α-synuclein, but not by A30P, E46K, and A53T forms of α-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools was markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that α-synuclein bound free 20:4n-6 (Kd of 3.7 μM), but did not bind 20:4n-6-CoA. These data suggest α-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that α-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum localized acyl-CoA synthetase activity, although mutants forms of α-synuclein fail to restore this activity. PMID:16734431

  12. Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG

    PubMed Central

    Mori, Giorgia; Chiarelli, Laurent R.; Esposito, Marta; Makarov, Vadim; Bellinzoni, Marco; Hartkoorn, Ruben C.; Degiacomi, Giulia; Boldrin, Francesca; Ekins, Sean; de Jesus Lopes Ribeiro, Ana Luisa; Marino, Leonardo B.; Centárová, Ivana; Svetlíková, Zuzana; Blaško, Jaroslav; Kazakova, Elena; Lepioshkin, Alexander; Barilone, Nathalie; Zanoni, Giuseppe; Porta, Alessio; Fondi, Marco; Fani, Renato; Baulard, Alain R.; Mikušová, Katarína; Alzari, Pedro M.; Manganelli, Riccardo; de Carvalho, Luiz Pedro S.; Riccardi, Giovanna; Cole, Stewart T.; Pasca, Maria Rosalia

    2015-01-01

    Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target. PMID:26097035

  13. Inhibition of gastric mucosal prostaglandin synthetase activity by mercaptomethylimidazole, an inducer of gastric acid secretion--plausible involvement of endogenous H2O2.

    PubMed

    Bhattacharjee, M; Chakraborty, T; Ganguly, C; Banerjee, R K

    1998-10-01

    We have reported earlier that mercaptomethylimidazole (MMI), an antithyroid drug of thionamide group, induces gastric acid secretion at least partially through the liberation of histamine, sensitive to cimetidine. Now, we show that the drug has a significant inhibitory effect on the cyclooxygenase and peroxidase activity of the prostaglandin (PG) synthetase of the gastric mucosal microsomal preparation. The effect can also be mimicked by low concentrations of H2O2. While studying the possible intracellular effect of MMI on acid secretion, a cell fraction (F3) enriched in parietal cell was isolated by controlled digestion of the mucosa with protease. This cell fraction is activated by MMI as measured by increased O2 consumption. The activation is sensitive to omeprazole, a proton-pump inhibitor, indicating that the activation is due to increased acid secretion by MMI. MMI was also found to directly inhibit the peroxidase activity of the F3 cell fraction and may thus increase the intracellular level of H2O2. The cyclooxygenase activity of the PG synthetase of the F3 cell fraction is also inhibited by MMI and the effect can be reproduced by low concentrations of H2O2. Both MMI and H2O2 can also inhibit the peroxidase activity of the PG synthetase. We suggest that in addition to the activation of the parietal cell by MMI possibly through endogenous H2O2, MMI induces acid secretion in vivo by inactivating the PG synthetase thereby inhibiting the biosynthesis of PG and removing its inhibitory influence on acid secretion so that the histamine released by MMI can stimulate acid secretion with maximum efficiency.

  14. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    PubMed

    Castro de Moura, Manuel; Miro, Francesc; Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-11-01

    Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

  15. Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner.

    PubMed

    Lee, Phil Young; Kho, Chang Won; Lee, Do Hee; Kang, Sunghyun; Kang, Seongman; Lee, Sang Chul; Park, Byoung Chul; Cho, Sayeon; Bae, Kwang-Hee; Park, Sung Goo

    2007-10-19

    Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.

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

    PubMed

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

    2014-08-01

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

  17. Atorvastatin prevents cell damage via modulation of oxidative stress, glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation.

    PubMed

    Vandresen-Filho, Samuel; Martins, Wagner C; Bertoldo, Daniela B; Mancini, Gianni; Herculano, Bruno A; de Bem, Andreza F; Tasca, Carla I

    2013-06-01

    Oxygen-glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.

  18. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    PubMed

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  19. Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens).

    PubMed

    Serapiglia, Michelle J; Minocha, Rakesh; Minocha, Subhash C

    2008-12-01

    We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount of N in the culture medium was reduced. When total N in the medium was increased, cell mass increased without significant changes in glutamine synthetase activity or polyamine concentration. Reductions in the amount of nitrate or total N in the culture medium resulted in increased accumulations of Ca, Mn and Zn in the cells, and K accumulation decreased in response to decreasing nitrate:ammonium ratios. The data indicate that changes in total N availability as well as the forms of N play important roles in the physiological responses of in-vitro-grown red spruce cells that mimic the observed responses of forest trees to soil N deficiency and N fertilization.

  20. Structure of the adenylylation domain of E. coli glutamine synthetase adenylyl transferase: evidence for gene duplication and evolution of a new active site.

    PubMed

    Xu, Yibin; Carr, Paul D; Vasudevan, Subhash G; Ollis, David L

    2010-02-26

    The X-ray structure of the C-terminal fragment, containing residues 449-946, of Escherichia coli glutamine synthetase adenylyl transferase (ATase) has been determined. ATase is part of the cascade that regulates the enzymatic activity of E. coli glutamine synthetase, a key component of the cell's machinery for the uptake of ammonia. It has two enzymatic activities, adenylyl removase (AR) and adenylyl transferase (AT), which are located in distinct catalytic domains that are separated by a regulatory (R) domain. We previously reported the three-dimensional structure of the AR domain (residues 1-440). The present structure contains both the R and AT domains. AR and AT share 24% sequence identity and also contain the beta-polymerase motif that is characteristic of many nucleotidylyl transferase enzymes. The structures overlap with an rmsd of 2.4 A when the superhelical R domain is omitted. A model for the complete ATase molecule is proposed, along with some refinements of domain boundaries. A rather more speculative model for the complex of ATase with glutamine synthetase and the nitrogen signal transduction protein PII is also presented.

  1. 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

  2. CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase

    PubMed Central

    He, Weiwei; Bai, Ge; Zhou, Huihao; Wei, Na; White, Nicholas M.; Lauer, Janelle; Liu, Huaqing; Shi, Yi; Dumitru, Calin Dan; Lettieri, Karen; Shubayev, Veronica; Jordanova, Albena; Guergueltcheva, Velina; Griffin, Patrick R.; Burgess, Robert W.; Pfaff, Samuel L.; Yang, Xiang-Lei

    2015-01-01

    Summary Selective neuronal loss is a hallmark of neurodegenerative diseases, which counter-intuitively are often caused by mutations in widely-expressed genes1. Charcot-Marie-Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies2,3. A subtype of the diseases—CMT2D—is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-tRNA synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons leading to deficits in distal motor function4. How mutations in GlyRS (GlyRSCMT2D) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRSCMT2D acquires a neomorphic binding activity that directly antagonizes an essential signaling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind the Neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRSCMT2D that antagonizes the VEGF/Nrp1 interaction, and indicate the VEGF/Nrp1 signaling axis is an actionable target for treating CMT2D. PMID:26503042

  3. 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.

  4. 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...

  5. Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

    PubMed Central

    Virág, L; Scott, G S; Cuzzocrea, S; Marmer, D; Salzman, A L; Szabó, C

    1998-01-01

    The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis

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

    PubMed

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

    2011-06-01

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

  7. Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis.

    PubMed Central

    Carrey, E A; Campbell, D G; Hardie, D G

    1985-01-01

    The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed. Images Fig. 1. Fig. 5. PMID:4092695

  8. Suppression of long chain acyl-CoA synthetase 3 decreases hepatic de novo fatty acid synthesis through decreased transcriptional activity.

    PubMed

    Bu, So Young; Mashek, Mara T; Mashek, Douglas G

    2009-10-30

    Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-gamma, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-alpha and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-(14)C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis.

  9. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation

    PubMed Central

    Grevengoed, Trisha J.; Cooper, Daniel E.; Young, Pamela A.; Ellis, Jessica M.; Coleman, Rosalind A.

    2015-01-01

    Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1T−/−) are virtually unable to oxidize fatty acids, glucose use increases 8-fold to compensate. This metabolic switch activates mechanistic target of rapamycin complex 1 (mTORC1), which initiates growth by increasing protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, Acsl1T−/− hearts contained 3 times more mitochondria with abnormal structure and displayed a 35–43% lower respiratory function. To study the effects of mTORC1 activation on mitochondrial structure and function, mTORC1 was inhibited by treating Acsl1T−/− and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1T−/− hearts, but did not improve ADP-stimulated oxygen consumption, which was likely caused by the 33–51% lower ATP synthase activity present in both vehicle- and rapamycin-treated Acsl1T−/− hearts. The turnover of microtubule associated protein light chain 3b in Acsl1T−/− hearts was 88% lower than controls, indicating a diminished rate of autophagy. Rapamycin treatment increased autophagy to a rate that was 3.1-fold higher than in controls, allowing the formation of autophagolysosomes and the clearance of damaged mitochondria. Thus, long-chain acyl-CoA synthetase isoform 1 (ACSL1) deficiency in the heart activated mTORC1, thereby inhibiting autophagy and increasing the number of damaged mitochondria.—Grevengoed, T. J., Cooper, D. E., Young, P. A., Ellis, J. M., Coleman, R. A. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation. PMID:26220174

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

    DTIC Science & Technology

    1980-09-01

    ammonia , enzyme, urea, brain, liver, kidney, gill, fish, salmon, herring, carp, catfish, hagfish, ratfish, dogfish, cod, stingray, Potarnotrygon,K... spleen , S; and spinal cord, SC. 4 Tissue activity, TA, is expressed in units per & tisue. Values below the lower limit of reliable detection, 1.5

  11. Long chain acyl-CoA synthetases and other acyl activating enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proper synthesis and breakdown of molecules containing carboxylic acids is a vital part of metabolism in all living organisms. Given the relatively inert chemical nature of many carboxylic acids, activation is a necessary step prior to use in the various anabolic and catabolic pathways that utilize...

  12. 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.

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

    PubMed Central

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

    1994-01-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7915611

  14. 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.

  15. 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

  16. Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

    PubMed

    Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

    2014-01-01

    Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

  17. A gene-type-specific enhancer regulates the carbamyl phosphate synthetase I promoter by cooperating with the proximal GAG activating element.

    PubMed Central

    Goping, I S; Lamontagne, S; Shore, G C; Nguyen, M

    1995-01-01

    The rat carbamyl phosphate synthetase I gene is expressed in two cell types: hepatocytes and epithelial cells of the intestinal mucosa. The proximal promoter contains a single activating element, GAG, two repressor elements (sites I and III) and an anti-repressor element (site II). Although these elements together exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity. Here we have identified a cell-type-specific enhancer that lies 10 kilobases upstream of the promoter. Unexpectedly, the enhancer also functioned in a gene-type-specific manner. The enhancer stimulated promoter activity exclusively through the proximal GAG element. Abrogation of GAG, either directly by mutation of GAG or indirectly by sites I and III repressors, abolished enhancer activation. Conversely, activation of the heterologous thymidine kinase promoter by the enhancer required the introduction of GAG. The requirement for GAG, therefore, functions to constrain the enhancer to a specific target promoter. PMID:7784176

  18. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

    SciTech Connect

    Buddha,M.; Crane, B.

    2005-01-01

    The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNATrp with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.

  19. Identification, Expression and IAA-Amide Synthetase Activity Analysis of Gretchen Hagen 3 in Papaya Fruit (Carica papaya L.) during Postharvest Process

    PubMed Central

    Liu, Kaidong; Wang, Jinxiang; Li, Haili; Zhong, Jundi; Feng, Shaoxian; Pan, Yaoliang; Yuan, Changchun

    2016-01-01

    Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya. PMID:27812360

  20. Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

    PubMed Central

    Ramirez, M; Wek, R C; Vazquez de Aldana, C R; Jackson, B M; Freeman, B; Hinnebusch, A G

    1992-01-01

    The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2. Images PMID:1448107

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

    PubMed

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

    1994-06-01

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

  2. Effects of Sheng-Mai injection on the PRPP synthetase activity in BFU-es and CFU-es from bone marrows of mice with benzene-induced aplastic anemia.

    PubMed

    Liu, L P; Liu, J F; Lu, Y Q

    2001-08-10

    160 Kunming mice were divided at random into 3 groups. Group 1: normal control (40 mice). Group 2: aplastic anemia (AA) control (60 mice); benzene inhalation was carried out for 2.5 months and sterilized normal saline was injected i.p. for another 6 weeks. Group 3: treated AA (60 mice); benzene was administered by inhalation in a similar manner, Sheng-Mai Injection (SMI) was administered i.p. for 6 weeks after the AA models were established. SMI is a famous Chinese traditional prescription of Panax ginseng C.A. Meyer (0.1 g/ml), Ophiopogon japonicus (Thunb.) Ker-Gawl (0.312 g/ml) and Fructus Schisandrae (0.158 g/ml). Activities of phosphoribosylpyrophosphate (PRPP) synthetase in BFU-Es and CFU-Es were estimated by ion pair reversed phase HPLC (IPrHPLC). Accompanying the sharp drop in counts of erythroid progenitor cells, the PRPP synthetase activity in CFU-Es of AA mice was reduced significantly (P<0.01), whereas there were no remarkable changes of this enzyme activity in their BFU-Es compared with the control group. Both the counts of erythroid progenitor cells and PRPP synthetase activity in CFU-Es returned nearly to normal levels following treatment with SMI of mice in Group 3 (P<0.01). Our results suggest that the attenuation of PRPP synthetase activity in peripheral erythrocytes of AA patients may originate from the weakening of activity of this enzyme in CFU-Es from their bone marrow. The impairment of PRPP formation would explain ATP depletion and disorders of energy metabolism in AA erythrocytes. SMI can distinctly increase the reduced quantity of erythroid progenitor cells and promote rapid restoration of PRPP synthetase activity in CFU-Es of AA mice.

  3. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    SciTech Connect

    Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

    2011-08-19

    Highlights: {yields} Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). {yields} Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. {yields} HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  4. 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

  5. A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence.

    PubMed

    Shoen, Carolyn M; DeStefano, Michelle S; Hager, Cynthia C; Tham, Kyi-Toe; Braunstein, Miriam; Allen, Alexandria D; Gates, Hiriam O; Cynamon, Michael H; Kernodle, Douglas S

    2013-01-11

    Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

  6. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-01-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective.

  7. Two tyrosine residues outside the editing active site in Giardia lamblia leucyl-tRNA synthetase are essential for the post-transfer editing.

    PubMed

    Zhou, Xiao-Long; Wang, En-Duo

    2009-08-28

    Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNA(Leu) synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNA(Leu) with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNA(Leu) in different conformations revealed that tRNA(Leu) 3' end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNA(Leu). Mutations on Y515 and Y520 also decreased tRNA(Leu) charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNA(Leu) 3' end with the CP1 domain in archaeal/eukaryotic LeuRS.

  8. [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.

  9. The rate of CO(2) assimilation controls the expression and activity of glutamine synthetase through sugar formation in sunflower (Helianthus annuus L) leaves.

    PubMed

    Larios, B; Aguera, E; Cabello, P; Maldonado, J M; de la Haba, P

    2004-01-01

    The expression and activity of glutamine synthetase (GS, EC 6.3.1.2) were examined in relation to the rate of CO2 assimilation in sunflower (Helianthus annuus L.) leaves. Intact plants were kept in the dark for 72 h and subsequently exposed to light under different atmospheric CO2 concentrations (100, 400 and 1200 microl l-1) for 6 h. The in vivo rates of net CO2 assimilation correlated with atmospheric CO2 concentrations. Stomatal conductances and transpiration rates remained largely unaffected by CO2 levels. Exposure of the plants to increasing CO2 concentrations in the light caused concomitant increases in the contents of starch and soluble sugars and a decrease in the nitrate content in leaves. Both cytosolic and chloroplastic (GS2) GS activities were higher at elevated CO2. A greater accumulation of GS2 mRNA was also observed under high CO2. Exogenous supply of sucrose to detached leaves greatly increased the levels of GS enzyme activity and of mRNA for chloroplastic GS in the dark. These results indicate that GS expression and activity in sunflower leaves are modulated by the rate of CO2 assimilation, and that photosynthesized sugars are presumably involved as regulatory metabolites.

  10. Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.

    PubMed

    Nakamoto, Terumasa; Miyanokoshi, Miki; Tanaka, Tomoaki; Wakasugi, Keisuke

    2016-04-20

    Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

  11. Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution

    PubMed Central

    Nakamoto, Terumasa; Miyanokoshi, Miki; Tanaka, Tomoaki; Wakasugi, Keisuke

    2016-01-01

    Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity. PMID:27094087

  12. Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

    PubMed Central

    Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

    2014-01-01

    SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

  13. 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.

  14. The Activation Mechanism of 2'-5'-Oligoadenylate Synthetase Gives New Insights Into OAS/cGAS Triggers of Innate Immunity.

    PubMed

    Lohöfener, Jan; Steinke, Nicola; Kay-Fedorov, Penelope; Baruch, Petra; Nikulin, Alexey; Tishchenko, Svetlana; Manstein, Dietmar J; Fedorov, Roman

    2015-05-05

    2'-5'-Oligoadenylate synthetases (OASs) produce the second messenger 2'-5'-oligoadenylate, which activates RNase L to induce an intrinsic antiviral state. We report on the crystal structures of catalytic intermediates of OAS1 including the OAS1·dsRNA complex without substrates, with a donor substrate, and with both donor and acceptor substrates. Combined with kinetic studies of point mutants and the previously published structure of the apo form of OAS1, the new data suggest a sequential mechanism of OAS activation and show the individual roles of each component. They reveal a dsRNA-mediated push-pull effect responsible for large conformational changes in OAS1, the catalytic role of the active site Mg(2+), and the structural basis for the 2'-specificity of product formation. Our data reveal similarities and differences in the activation mechanisms of members of the OAS/cyclic GMP-AMP synthase family of innate immune sensors. In particular, they show how helix 3103-α5 blocks the synthesis of cyclic dinucleotides by OAS1.

  15. Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Deniel, Nicolas; Husson, Annie

    2006-06-01

    The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.

  16. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    PubMed

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

  17. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    PubMed

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process.

  18. Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

    PubMed

    Dal-Cim, Tharine; Martins, Wagner C; Thomaz, Daniel T; Coelho, Victor; Poluceno, Gabriela Godoy; Lanznaster, Débora; Vandresen-Filho, Samuel; Tasca, Carla I

    2016-05-01

    Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.

  19. Long chain acyl-CoA synthetase-3 is a molecular target for peroxisome proliferator-activated receptor delta in HepG2 hepatoma cells.

    PubMed

    Cao, Aiqin; Li, Hai; Zhou, Yue; Wu, Minhao; Liu, Jingwen

    2010-05-28

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

  20. A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli.

    PubMed

    Jiang, Peng; Ninfa, Alexander J

    2011-12-20

    Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.

  1. Effect of ploidy levels on the activities of delta(1)-pyrroline-5-carboxylate synthetase, superoxide dismutase and peroxidase in Cenchrus species grown under water stress.

    PubMed

    Chandra, Amaresh; Dubey, Archana

    2010-01-01

    The effects of ploidy levels on the activities of delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), superoxide dismutase (SOD; EC 1.15.1.1) and guaiacol peroxidase (POX; EC 1.11.1.7), as well as malondialdehyde (MDA) and proline contents were studied in two months old plants of Cenchrus species. The Cenchrus species represent three ploidy levels: diploid, tetraploid, hexaploid and two life spans: annual and perennial. Plants were subjected to water stress for 2, 4, 6 and 8 d by withholding water under glasshouse conditions. Although the levels of proline increased with the magnitude of water stress, the P5CS activity did not show a corresponding increase in all species. Peroxidase and superoxide dismutase activities showed an increase or steady state in the early phase of drought and then declined with the further increase in the magnitude of water stress, indicating differing behaviors of species towards drought tolerance. Under drought, diploid Cenchrus species had a higher POX activity, MDA accumulation and lower proline content than tetraploid species. Lower POX and higher P5CS activities and proline contents, however, were observed in hexaploid and tetraploid species. Taken together, our findings suggest that diploid species have a less efficient antioxidant system to scavenge reactive oxygen species than tetra and hexaploid Cenchrus. This may result in a corresponding variability in growth and persistence under natural grasslands. The study also paves the way for investigations on the molecular events associated with drought in Cenchrus species differing in ploidy and life span.

  2. Inhibition of cystathionine β-synthetase suppresses sodium channel activities of dorsal root ganglion neurons of rats with lumbar disc herniation

    PubMed Central

    Yan, Jun; Hu, Shufen; Zou, Kang; Xu, Min; Wang, Qianliang; Miao, Xiuhua; Yu, Shan Ping; Xu, Guang-Yin

    2016-01-01

    The pathogenesis of pain in lumbar disc herniation (LDH) remains poorly understood. We have recently demonstrated that voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons were sensitized in a rat model of LDH. However, the detailed molecular mechanism for sensitization of VGSCs remains largely unknown. This study was designed to examine roles of the endogenous hydrogen sulfide synthesizing enzyme cystathionine β-synthetase (CBS) in sensitization of VGSCs in a previously validated rat model of LDH. Here we showed that inhibition of CBS activity by O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) significantly attenuated pain hypersensitivity in LDH rats. Administration of AOAA also reduced neuronal hyperexcitability, suppressed the sodium current density, and right-shifted the V1/2 of the inactivation curve, of hindpaw innervating DRG neurons, which is retrogradely labeled by DiI. In vitro incubation of AOAA did not alter the excitability of acutely isolated DRG neurons. Furthermore, CBS was colocalized with NaV1.7 and NaV1.8 in hindpaw-innervating DRG neurons. Treatment of AOAA markedly suppressed expression of NaV1.7 and NaV1.8 in DRGs of LDH rats. These data suggest that targeting the CBS-H2S signaling at the DRG level might represent a novel therapeutic strategy for chronic pain relief in patients with LDH. PMID:27905525

  3. Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet.

    PubMed

    Mashek, Douglas G; Li, Lei O; Coleman, Rosalind A

    2006-09-01

    Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms.

  4. Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

    PubMed Central

    Christoffels, Vincent M.; Grange, Thierry; Kaestner, Klaus H.; Cole, Timothy J.; Darlington, Gretchen J.; Croniger, Colleen M.; Lamers, Wouter H.

    1998-01-01

    A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPα-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPβ knockout mice and in HNF3α and -γ double-knockout mice. (The role of HNFβ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway. PMID:9774647

  5. Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP).

    PubMed

    Konno, Michiko; Sumida, Tomomi; Uchikawa, Emiko; Mori, Yukie; Yanagisawa, Tatsuo; Sekine, Shun-ichi; Yokoyama, Shigeyuki; Yokoyama, Shigeuki

    2009-09-01

    The ATP-pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln-tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyrophosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3'-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg-tRNA synthetase from Pyrococcus horikoshii, tRNA(Arg)(CCU) and an ATP analog with Rfactor = 0.213 (Rfree = 0.253) at 2.0 A resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2'-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP --> Arg-AMP + pyrophosphate) and pyrophosphorolysis of Arg-AMP (Arg-AMP + pyrophosphate --> Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-tRNA + AMP --> Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (-NH-C+(NH2)2 or -COOH) of Arg and Glu assists the aminoacyl transfer from the 2'-OH group of tRNA to the phosphate group of AMP at high pH.

  6. Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity1[OPEN

    PubMed Central

    Pedersen, Carsten

    2016-01-01

    Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of the high-affinity low-capacity isoform Gln-1;1 was reduced. The expression of Gln-1;3 did not respond to ammonium treatment while Gln-1;4 and Gln-1;5 isogenes in all cases were expressed at a very low level. Gln-2 was highly expressed in shoots but only at a very low level in roots. To investigate the specific functions of the two isogenes Gln-1;1 and Gln-1;2 in shoots for ammonium detoxification, single and double knock-out mutants were grown under standard N supply or with high ammonium provision. Phenotypes of the single mutant gln1;1 were similar to the wild type, while growth of the gln1;2 single mutant and the gln1;1:gln1;2 double mutant was significantly impaired irrespective of N regime. GS1 activity was significantly reduced in both gln1;2 and gln1;1:gln1;2. Along with this, the ammonium content increased while that of Gln decreased, showing that Gln-1;2 was essential for ammonium assimilation and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots. PMID:27231101

  7. The CP2 domain of leucyl-tRNA synthetase is crucial for amino acid activation and post-transfer editing.

    PubMed

    Zhou, Xiao-Long; Zhu, Bin; Wang, En-Duo

    2008-12-26

    Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.

  8. Glutamine accumulation and up-regulation of glutamine synthetase activity in the swamp eel, Monopterus albus (Zuiew), exposed to brackish water.

    PubMed

    Tok, Chia Y; Chew, Shit F; Peh, Wendy Y X; Loong, Ai M; Wong, Wai P; Ip, Yuen K

    2009-05-01

    The swamp eel, Monopterus albus, is an air-breathing teleost which typically lives in freshwater but can also be found in estuaries, where it has to deal with ambient salinity fluctuations. Unlike other teleosts, its gills are highly degenerate. Hence, it may have uncommon osmoregulatory adaptations, but no information is available on its osmoregulatory capacity and mechanisms at present. In this study M. albus was exposed to a 5 day progressive increase in salinity from freshwater (1 per thousand) to brackish water (25 per thousand) and subsequently kept in 25 per thousand water for a total of 4 days. The results indicate that M. albus switched from hyperosmotic hyperionic regulation in freshwater to a combination of osmoconforming and hypoosmotic hypoionic regulation in 25 per thousand water. Exposure to 25 per thousand water resulted in relatively large increases in plasma osmolality, [Na(+)] and [Cl(-)]. Consequently, fish exposed to 25 per thousand water had to undergo cell volume regulation through accumulation of organic osmolytes and inorganic ions. Increases in tissue free amino acid content were apparently the result of increased protein degradation, decreased amino acid catabolism, and increased synthesis of certain non-essential amino acids. Here we report for the first time that glutamine is the major organic osmolyte in M. albus. Glutamine content increased to a phenomenal level of > 12 micromol g(-1) and > 30 micromol g(-1) in the muscle and liver, respectively, of fish exposed to 25 per thousand water. There were significant increases in glutamine synthetase (GS) activity in muscle and liver of these fish. In addition, exposure to 25 per thousand water for 4 days led to significant increases in GS protein abundance in both muscle and liver, indicating that increases in the expression of GS mRNA could have occurred.

  9. 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

  10. Activation of a Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic Nonribosomal Peptide Synthetase Gene Cluster ▿ †

    PubMed Central

    Bergmann, Sebastian; Funk, Alexander N.; Scherlach, Kirstin; Schroeckh, Volker; Shelest, Ekaterina; Horn, Uwe; Hertweck, Christian; Brakhage, Axel A.

    2010-01-01

    Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion

  11. Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.

    PubMed

    Lamech, Lilian T; Mallam, Anna L; Lambowitz, Alan M

    2014-12-01

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was "fixed" by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

  12. Part I. Cobalt thiolate complexes modeling the active site of cobalt nitrile hydratase. Part II. Formation of inorganic nanoparticles on protein scaffolding in Escherichia coli glutamine synthetase

    NASA Astrophysics Data System (ADS)

    Kung, Irene Yuk Man

    Part I. A series of novel cobalt dithiolate complexes with mixed imine/amine ligand systems is presented here as electronic and structural models for the active site in the bacterial enzyme class, nitrile hydratase (NHase). Pentadentate cobalt(II) complexes with S2N 3 ligand environments are first studied as precursors to the more relevant cobalt(III) complexes. Adjustment of the backbone length by removal of a methylene group increases the reactivity of the system; whereas reduction of the two backbone imine bonds to allow free rotation about those bonds may decrease reactivity. Reactivity change due to the replacement of the backbone amine proton with a more sterically challenging methyl group is not yet clear. Upon oxidation, the monocationic pentadentate cobalt(III) complex, 1b, shows promising reactivity similar to that of NHase. The metal's open coordination site allows reversible binding of the endogenous, monoanionic ligands, N 3- and NCS-. Oxygenation of the thiolate sulfur atoms by exposure to O2 and H2O 2 produces sulfenate and sulfinate ligands in complex 8, which resembles the crystal structure of "deactivated" Fe NHase. However, its lack of reactivity argues against the oxygenated enzyme structure as the active form. Six-coordinate cobalt(III) complexes with S2N4 amine/amine ligand systems are also presented as analogues of previously reported iron(III) compounds, which mimic the spectroscopic properties of Fe NHase. The cobalt complexes do not seem to similarly model Co NHase. However, the S = 0 cobalt(III) center can be spectroscopically silent and difficult to detect, making comparison with synthetic models using common techniques hard. Part II. Dodecameric Escherichia coli glutamine synthetase mutant, E165C, stacks along its six-fold axis to produce tubular nanostructures in the presence of some divalent metal ions, as does the wild type enzyme. The centrally located, engineered Cys-165 residues appear to bind to various species and may serve as

  13. 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)

  14. 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 ...

  15. Rosiglitazone Inhibits Acyl-CoA Synthetase Activity and Fatty Acid Partitioning to Diacylglycerol and Triacylglycerol via a Peroxisome Proliferator–Activated Receptor-γ–Independent Mechanism in Human Arterial Smooth Muscle Cells and Macrophages

    PubMed Central

    Askari, Bardia; Kanter, Jenny E.; Sherrid, Ashley M.; Golej, Deidre L.; Bender, Andrew T.; Liu, Joey; Hsueh, Willa A.; Beavo, Joseph A.; Coleman, Rosalind A.; Bornfeldt, Karin E.

    2010-01-01

    Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator–activated receptor (PPAR)-γ, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-γ–independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-γ–independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis. PMID:17259370

  16. 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.

  17. Comparison of ent-kaurene synthetase A and B activities in cell-free extracts from young tomato fruits of wild-type, and dwarf gib-1 and gib-3 tomato plants

    SciTech Connect

    Bensen, R.J.; Zeevaart, J.A.D. )

    1990-05-01

    The non-allelic gib-1 and gib-3 tomato (Lycopersion esculentum Mill.) mutants are gibberellin deficient and exhibit a dwarfed gowth habit. Previous work has shown that this dwarfed growth pattern can be reversed by the application of a number of gibberellins and their precursors, including ent-kaurene. This indicates that the mutants are blocked in gibberellin biosynthesis at a step prior to ent-kaurene metabolism. The normal accumulation of carotenoids observed in these mutants suggests a fully functional isoprenoid pathway. Ent-kaurene is synthesized from the isoprenoid geranylgeranyl pyrophosphate in a two-step process with copalyl pyrophosphate as an interemediate. In vitro assays using young fruit extracts from gib-1 plants, indicated a reduced ability for synthesis of copalyl pyrophosphate from geranylgeranyl pyrophosphate, and thus a reduced ent-kaurene synthetase A activity. Similarly, gib-3 extracts demonstrated a reduced ability to synthesize ent-kaurene from copalyl pyrophosphate, and thus a reduced ent-kaurene synthetase B activity. These results establish the site of the mutation in gib-1 and gib-3 tomatoes as being in the enzymatic conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and copalyl pyrophosphate to ent-kaurene, respectively.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

    PubMed

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

    2016-03-04

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

  3. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter*

    PubMed Central

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

    2016-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5′-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript. PMID:26728456

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Modulating the synthetase activity of penicillin G acylase in organic media by addition of N-methylimidazole: using vinyl acetate as activated acyl donor.

    PubMed

    Liu, Bokai; Wu, Qi; Lv, Deshui; Lin, Xianfu

    2011-05-20

    This paper reported the modulation of enzyme activity by organic small molecule. The esterification activity of Penicillin G acylase (PGA) was improved more than 70-fold by the addition of 10% N-methylimidazole. Some control experiments have been designed to demonstrate the catalytic specificity of PGA. The structure and the amount of additive were optimized to improve the product yield. The influence of N-methylimidazole on the PGA conformation was investigated by FTIR and autodock simulation. Seven substrates were used to evaluate the effect of structure on the PGA-catalyzed transesterification. A series of products were successfully synthesized with the yield ranged from 56% to 84% and PGA showed specific recognition on the substrate with phenyl group in the presence of 10% N-methylimidazole.

  14. Activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA.

    PubMed Central

    Han, Jian-Qiu; Barton, David J

    2002-01-01

    Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA. PMID:11991644

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

    PubMed Central

    Waterson, Robert M.; Konigsberg, William H.

    1974-01-01

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

  16. 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

  17. 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

  18. 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.

  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. 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. 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.

  2. 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.

  3. 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

  4. 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

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

    PubMed

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

    1995-08-01

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

  6. 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

  7. 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.

  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. 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 ...

  10. 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

  11. 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.

  12. 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.

  13. Mitochondrial aminoacyl-tRNA synthetases in human disease.

    PubMed

    Konovalova, Svetlana; Tyynismaa, Henna

    2013-04-01

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

  14. Light induces changes in activities of Na+/K+-ATPase, H+/K+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam, Tridacna squamosa

    PubMed Central

    Ip, Yuen K.; Ching, Biyun; Hiong, Kum C.; Choo, Celine Y. L.; Boo, Mel V.; Wong, Wai P.; Chew, Shit F.

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH+4 or H+, and activities of enzymes involved in converting NH+4 to glutamate/glutamine in inner mantle, outer mantle, and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH+4 in substitution for K+ to activate Na+/K+-ATPase (NKA), manifested as a significant increase in the Na+/NH+4-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H+ released from CaCO3 deposition could react with NH3 to form NH+4 in the extrapallial fluid, and NH+4 could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH+4 and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM)-sensitive-V-H+-ATPase (VATPase) in the inner mantle, and significant increases in the Na+/K+-activated-NKA, H+/NH+4-activated-H+/K+-ATPase, and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na+ homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH+4 from the environment. This is the first report on light having direct enhancing effects on activities of certain transporters

  15. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase.

    PubMed

    Faesen, Alex C; Dirac, Annette M G; Shanmugham, Anitha; Ovaa, Huib; Perrakis, Anastassis; Sixma, Titia K

    2011-10-07

    The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state.

  16. 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

  17. Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase-Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase.

    PubMed

    Sahu, Divya; Gupta, Sounak; Hau, Andrew M; Nakashima, Kazufumi; Leivo, Mariah Z; Searles, Stephen C; Elson, Paul; Bomalaski, John S; Casteel, Darren E; Boss, Gerry R; Hansel, Donna E

    2016-12-09

    Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

  18. 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

  19. Acetyl-CoA synthetase is activated as part of the PDH-bypass in the oleaginous green alga Chlorella desiccata

    PubMed Central

    Avidan, Omri; Pick, Uri

    2015-01-01

    In a recent study, it has been shown that biosynthesis of triacylglycerol (TAG) in the oleaginous green alga Chlorella desiccata is preceded by a large increase in acetyl-coenzyme A (Ac-CoA) levels and by upregulation of plastidic pyruvate dehydrogenase (ptPDH). It was proposed that the capacity to accumulate high TAG critically depends on enhanced production of Ac-CoA. In this study, two alternative Ac-CoA producers—plastidic Ac-CoA synthase (ptACS) and ATP citrate lyase (ACL)—are shown to be upregulated prior to TAG accumulation under nitrogen deprivation in the oleaginous species C. desiccata, but not in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Measurements of endogenous acetate production and of radiolabelled acetate incorporation into lipids are consistent with the upregulation of ptACS, but suggest that its contribution to the overall TAG biosynthesis is negligible. Induction of ACS and production of endogenous acetate are correlated with activation of alcohol dehydrogenase, suggesting that the upregulation of ptACS is associated with activation of PDH-bypass in C. desiccata. It is proposed that activation of the PDH-bypass in C. desiccata is needed to enable a high rate of lipid biosynthesis under nitrogen deprivation by controlling the level of pyruvate reaching ptPHD and/or mtPDH. This may be an important parameter for massive TAG accumulation in microalgae. PMID:26357883

  20. 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

  1. 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.

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

    PubMed Central

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

    1992-01-01

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

  3. 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

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

    PubMed

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

    2011-01-01

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

  5. The 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

  6. 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.

  7. 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.

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

    SciTech Connect

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

    1987-01-27

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

  9. 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.

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

    PubMed

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

    2012-12-01

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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.].

  16. 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)

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. A Rare Case of Short-Chain Acyl-COA Dehydrogenase Deficiency: The Apparent Rarity of the Disorder Results in Under Diagnosis.

    PubMed

    Reddy, G Shilpa; Sujatha, M

    2011-07-01

    Short-chain acyl-CoA dehydrogenase (ACAD) deficiency is an extremely rare inherited mitochondrial disorder of fat metabolism. This belongs to a group of diseases known as fatty acid oxidation disorders. Screening programmes have provided evidence that all the fatty acid oxidation disorders combined are among the most common inborn errors of metabolism. Mitochondrial beta oxidation of fatty acids is an essential energy producing pathway. It is a particularly important pathway during prolonged periods of starvation and during periods of reduced caloric intake due to gastrointestinal illness or increased energy expenditure during febrile illness. The most common presentation is an acute episode of life threatening coma and hypoglycemia induced by a period of fasting due to defective hepatic ketogenesis. Here, the case of a 4 month old female patient who had seizures since the third day of her birth and persistent hypoglycemia is described. She was born to parents of second degree consanguinity after 10 years of infertility treatment. There was history of delayed cry after birth. Metabolic screening for TSH, galactosemia, 17-OHP, G6PD, cystic fibrosis, biotinidase were normal. Tandem mass spectrometric (TMS) screening for blood amino acids, organic acids, fatty acids showed elevated butyryl carnitine (C4) as 3.40 μmol/L (normal <2.00 μmol/L), hexanoyl carnitine (C6) as 0.92 μmol/L (normal <0.72 μmol/L), C4/C3 as 2.93 μmol/L (normal <1.18 μmol/L). The child was started immediately on carnitor syrup (carnitine) 1/2 ml twice daily. Limitation of fasting stress and dietary fat was advised. Baby responded well by gaining weight and seizures were controlled. Until now, less than 25 patients have been reported worldwide. The limited number of patients diagnosed until now is due to the rarity of the disorder resulting in under diagnosis.

  4. 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

  5. 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.

  6. The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker.

    PubMed

    Keen, M J; Hale, C

    1995-01-01

    A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H(*) were adapted to enable them to grow serum-free in the absence of cholesterol and β-cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10(6) cells ml(-1) producing 76.6 mg l(-1) of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.

  7. Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

    PubMed

    Wang, Xiaoyue; Wang, Guanglu; Li, Xinli; Fu, Jing; Chen, Tao; Wang, Zhiwen; Zhao, Xueming

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

  8. 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.

  9. 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.,

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  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. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Total glutamine synthetase levels in cerebrospinal fluid of Alzheimer's disease patients are unchanged.

    PubMed

    Timmer, Nienke M; Herbert, Megan K; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2015-03-01

    Decreased cerebral protein and activity levels of glutamine synthetase (GS) have been reported for Alzheimer's disease (AD) patients. Using a recently established method, we quantified total GS levels in cerebrospinal fluid (CSF) from AD patients and control subjects. Furthermore, we investigated if total GS levels in CSF could differentiate AD from frontotemperal dementia and dementia with Lewy bodies patients. As we found no significantly altered total GS levels in any of the patient groups compared with control subjects, we conclude that levels of total GS in CSF have no diagnostic value for AD, dementia with Lewy bodies, or frontotemperal dementia.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Conversion of rat liver S-adenosyl-L-methionine synthetase from high-Mr form to low-Mr form by LiBr.

    PubMed

    Cabrero, C; Alemany, S

    1988-02-10

    Rat liver S-adenosyl-L-methionine synthetase exists in two forms which are, respectively, a dimer and a tetramer of an Mr 48,500 subunit. The high-molecular-mass form is converted into the low-molecular-mass form by incubation with 1.4 M LiBr. The kinetic properties of the low-molecular-mass form obtained by LiBr treatment are the same as those obtained with the low-molecular-mass S-adenosyl-L-methionine synthetase form purified from rat liver cytosol. These results demonstrate that the differences in specific activity and regulatory properties of the high-molecular-mass and the low-molecular-mass S-adenosyl-L-methionine synthetase forms are due to their different polymeric states.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  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. 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

  13. 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.

  14. Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N- myristoylation

    PubMed Central

    1992-01-01

    Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N- myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp. PMID:1572893

  15. 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.

  16. 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

  17. 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

  18. Computational Sampling and Simulation Based Assessment of Novel Mycobacterium tuberculosis Glutamine Synthetase Inhibitors: Study Involving Structure Based Drug Design and Free Energy Perturbation.

    PubMed

    Suryadevara, Priyanka; Yogeeswari, Perumal; Soni, Vijay; Devi, Parthiban Brindha; Nandicoori, Vinay Kumar; Sriram, Dharmarajan

    2016-01-01

    The highly persistent nature of Mycobacterium tuberculosis can be attributed to its lipophilic cell wall which acts as a major barrier in the process of drug discovery against tuberculosis. Glutamine synthetase plays a major role in nitrogen metabolism and cell wall biosynthesis of pathogenic mycobacteria. The current review focuses on the structural and functional aspects of Mtb glutamine synthetase and an overview of its reported inhibitors till date. Also in the present study, we employed a computational structure based drug design protocol for identifying novel inhibitors against Mtb glutamine synthetase (MtbGS). A total of 12 hits were identified based on e-pharmacophore related search and virtual screening, which were further tested for their in vitro MtbGS inhibitory activity. Three compounds (compound 6, 1 and 12) were found with IC50 less than 5 µM, of which compound 6 being top active with IC50 of 2.124 µM. Differential scanning fluorimetry studies were employed so as to measure the thermal stability of the protein complexed with the most active compound. Also the protein complexes with top three active compounds were subjected for molecular dynamics simulations to study their binding pattern and stabilization effect. The solvation free energies were also determined for these compounds, undertaking free energy perturbation studies, which can be used further for lead optimization in the process of anti-tubercular drug discovery targeting Mtb glutamine synthetase.

  19. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  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. Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.

    PubMed

    Suo, Z; Walsh, C T; Miller, D A

    1999-10-19

    The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic

  4. 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

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

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

    PubMed

    Kalkman, Hans O

    2011-06-01

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

  17. 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.

  18. MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

    PubMed Central

    Murdeshwar, Maya S.

    2012-01-01

    In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme RelMsm. This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The relMsm knockout strain of M. smegmatis (ΔrelMsm) is expected to show a (p)ppGpp null [(p)ppGpp0] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRelMsm in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response. PMID:22636779

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Acetylation of prostaglandin synthetase by aspirin. Purification and properties of the acetylated protein from sheep vesicular gland.

    PubMed

    Roth, G J; Stanford, N; Jacobs, J W; Majerus, P W

    1977-09-20

    We previously presented evidence that aspirin (acetylsalicylic acid) inhibits prostaglandin synthetase by acetylating and active site of the enzyme. In the current work, we have labeled the enzyme from an aceton-pentane powder of sheep vesicular gland using [acetyl-3H]aspirin and purified the [3H]acetyl-protein to near homogeneity. The final preparation contains protein of a single molecular weight (85 000) and an amino-terminal sequence of Asp-Ala-Gly-Arg-Ala. The [3H]acetyl-protein contained 0.5 mol of acetyl residues per mol of protein based on amino acid composition but only a single sequence was found.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Application of a Propionyl Coenzyme A Synthetase for Poly(3-Hydroxypropionate-co-3-Hydroxybutyrate) Accumulation in Recombinant Escherichia coli

    PubMed Central

    Valentin, Henry E.; Mitsky, Timothy A.; Mahadeo, Debbie A.; Tran, Minhtien; Gruys, Kenneth J.

    2000-01-01

    The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381–1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a Km of 50 μM and a specific activity of 120 μmol · min−1 · mg−1 were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590–6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291–299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity. PMID:11097899

  9. Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.

    PubMed Central

    Lamour, V; Quevillon, S; Diriong, S; N'Guyen, V C; Lipinski, M; Mirande, M

    1994-01-01

    An important step ensuring the fidelity in protein biosynthesis is the aminoacylation of tRNAs by aminoacyl-tRNA synthetases. The accuracy of this process rests on a family of 20 enzymes, one for each amino acid. One exception is the formation of Gln-tRNA(Gln) that can be accomplished by two different pathways: aminoacylation of tRNA(Gln) with Gln by glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) or transamidation of Glu from Glu-tRNA(Gln) mischarged by glutamyl-tRNA synthetase (GluRS; EC 6.1.1.17). The latter pathway is widespread among bacteria and organelles that, accordingly, lack GlnRS. However, some bacterial species, such as Escherichia coli, do possess a GlnRS activity, which is responsible for Gln-tRNA(Gln) formation. In the cytoplasm of eukaryotic cells, both GluRS and GlnRS activities can be detected. To gain more insight into the evolutionary relationship between GluRS and GlnRS enzyme species, we have now isolated and characterized a human cDNA encoding GlnRS. The deduced amino acid sequence shows a strong similarity with other known GlnRSs and with eukaryotic GluRSs. A molecular phylogenetic analysis was conducted on the 14 GlxRS (GluRS or GlnRS) sequences available to date. Our data suggest that bacterial GlnRS has a eukaryotic origin and was acquired by a mechanism of horizontal gene transfer. Images PMID:8078941

  10. Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

    PubMed Central

    Kumar, Sandeep; Das, Mom; Hadad, Christopher M.; Musier-Forsyth, Karin

    2012-01-01

    Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNAPro. Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNAPro, whereas Cys-tRNAPro is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNAPro deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site, as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2′-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein’s conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis. PMID:22458656

  11. Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W

    PubMed Central

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J.; Legrand, Michel

    2010-01-01

    The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors. PMID:21193572

  12. 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

  13. 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

  14. 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.

  15. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    PubMed

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

  16. Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics.

    PubMed

    Hartwig, Silvia; Dovengerds, Christine; Herrmann, Christian; Hovemann, Bernhard T

    2014-11-01

    Drosophila Ebony is a β-alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases (NRPSs), which typically consist of at least an adenylation, a peptidyl carrier protein and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to β-alanyl conjugates. If the metabolization of the neurotransmitter histamine to β-alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPSs are in general slowly acting multi-modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remains a key question for understanding its role in vision. To quantitatively analyze the reaction kinetics, we used phosphopantetheinylated holo-Ebony prepared from Baculovirus infected Sf9 cells. Kinetic parameters for the loading reaction, e.g. the formation of β-alanyl-Ebony thioester, complied with those of slow NRPSs. In contrast, single-turnover analysis of the last reaction step, peptide bond formation between pre-activated β-alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This biphasic nature of activity identifies Ebony as a novel type of NRPS related molecule that combines a slow amino acid activation phase with a very fast product formation step.

  17. Structure-guided expansion of the substrate range of methylmalonyl coenzyme A synthetase (MatB) of Rhodopseudomonas palustris.

    PubMed

    Crosby, Heidi A; Rank, Katherine C; Rayment, Ivan; Escalante-Semerena, Jorge C

    2012-09-01

    Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as Escherichia coli often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium Rhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report the apo (1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of RpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site of RpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.

  18. Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

    PubMed

    Apfel, C M; Takács, B; Fountoulakis, M; Stieger, M; Keck, W

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

  19. The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

    PubMed Central

    Sissler, M; Giegé, R; Florentz, C

    1998-01-01

    Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system. PMID:9622124

  20. Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential uppS Gene

    PubMed Central

    Apfel, Christian M.; Takács, Béla; Fountoulakis, Michael; Stieger, Martin; Keck, Wolfgang

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E,E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6. PMID:9882662

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

    PubMed

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

    2004-10-01

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

  2. 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.

  3. 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.

  4. The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase.

    PubMed Central

    Ueda, T; Yotsumoto, Y; Ikeda, K; Watanabe, K

    1992-01-01

    Recognition sites of bovine mitochondrial serine tRNA specific for condons AGY [tRNA(Ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(Ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase. PMID:1375735

  5. L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus.

    PubMed

    Shiau, Chia-Yang; Liu, Yu-Tien

    2002-04-12

    L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

  6. Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens.

    PubMed

    Li, Li; Palencia, Andrés; Lukk, Tiit; Li, Zhi; Luthey-Schulten, Zaida A; Cusack, Stephen; Martinis, Susan A; Boniecki, Michal T

    2013-03-05

    Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA(Leu), resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.

  7. Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis.

    PubMed

    Jain, Vitul; Yogavel, Manickam; Oshima, Yoshiteru; Kikuchi, Haruhisa; Touquet, Bastien; Hakimi, Mohamed-Ali; Sharma, Amit

    2015-05-05

    The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

  8. Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells.

    PubMed Central

    Aslanian, A M; Kilberg, M S

    2001-01-01

    Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12 h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine. PMID:11485552

  9. Structure of Trypanosoma brucei glutathione synthetase: Domain and loop alterations in the catalytic cycle of a highly conserved enzyme

    PubMed Central

    Fyfe, Paul K.; Alphey, Magnus S.; Hunter, William N.

    2010-01-01

    Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-γ-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery. PMID:20045436

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Mammalian folylpoly-. gamma. -glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis

    SciTech Connect

    Cook, J.D.; Cichowicz, D.J.; George, S.; Lawler, Ann; Shane, B.

    1987-01-27

    The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B/sub 12/ status, can be mimicked in vitro by using purified enzyme. (/sup 3/H)Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B/sub 12/ deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly.

  15. Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy

    PubMed Central

    Gruenbaum, Shaun E.; Wang, Helen; Zaveri, Hitten P.; Tang, Amber B.; Lee, Tih-Shih W.; Eid, Tore; Dhaher, Roni

    2015-01-01

    The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague–Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n = 12)] or phosphate buffered saline [PBS (n = 7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21 days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21 days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p < 0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE. PMID:26262937

  16. A Novel Tool for Studying Auxin-Metabolism: The Inhibition of Grapevine Indole-3-Acetic Acid-Amido Synthetases by a Reaction Intermediate Analogue

    PubMed Central

    Böttcher, Christine; Dennis, Eric G.; Booker, Grant W.; Polyak, Steven W.; Boss, Paul K.; Davies, Christopher

    2012-01-01

    An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA) by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-5′-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP) mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with Ki-values 17-68-fold lower than the respective Km-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5–20 µM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function. PMID:22649546

  17. Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy.

    PubMed

    Gruenbaum, Shaun E; Wang, Helen; Zaveri, Hitten P; Tang, Amber B; Lee, Tih-Shih W; Eid, Tore; Dhaher, Roni

    2015-10-01

    The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.

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

    PubMed

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

    1983-04-01

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

  20. A Master Switch Couples Mg2+-Assisted Catalysis to Domain Motion in B. stearothermophilus Tryptophanyl-tRNA Synthetase

    PubMed Central

    Weinreb, Violetta; Li, Li; Carter, Charles W.

    2011-01-01

    Summary We demonstrate how Tryptophanyl-tRNA synthetase (TrpRS) uses conformation-dependent Mg2+ activation to couple catalysis of tryptophan activation to specific, functional domain movements. Rate acceleration by Mg2+ requires ~ -6.0 kcal/mole in protein•Mg2+ interaction energy, none of which arises from the active site. A highly cooperative interaction between Mg2+ and four residues from a remote, conserved motif that mediates the shear of domain movement: (i) destabilizes the pre-transition state conformation, thereby (ii) inducing the Mg2+ to stabilize the transition state for kcat by ~ -5.0 kcal/mole. Cooperative, long-range conformational effects on the metal therefore convert an inactive Mg2+ coordination into one that can stabilize the transition state if, and only if, domain motion occurs. Transient, conformation-dependent Mg2+ activation, analogous to the escapement in mechanical clocks, explains vectorial coupling. PMID:22244762

  1. Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli.

    PubMed

    Okano, Hiroyuki; Hwa, Terence; Lenz, Peter; Yan, Dalai

    2010-12-03

    Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR(-)/AT(+) mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR(-) mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.

  2. 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.

  3. 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

  4. 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).

  5. 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

  6. 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

  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. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site.

    PubMed

    Li, Sheng; Lu, Yongcheng; Peng, Baozhen; Ding, Jianping

    2007-01-01

    PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

  18. Biosynthesis of amphi-enterobactin siderophores by Vibrio harveyi BAA-1116: identification of a bifunctional nonribosomal peptide synthetase condensation domain.

    PubMed

    Zane, Hannah K; Naka, Hiroaki; Rosconi, Federico; Sandy, Moriah; Haygood, Margo G; Butler, Alison

    2014-04-16

    The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.

  19. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    PubMed

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

  20. Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.

    PubMed

    Ray, Partho Sarothi; Fox, Paul L

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering "missing links" in the tree of life.

  1. Origin and Evolution of Glutamyl-prolyl tRNA Synthetase WHEP Domains Reveal Evolutionary Relationships within Holozoa

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. PMID:24968216

  2. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    SciTech Connect

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  3. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

    PubMed Central

    Palencia, Andrés; Li, Xianfeng; Bu, Wei; Choi, Wai; Ding, Charles Z.; Easom, Eric E.; Feng, Lisa; Hernandez, Vincent; Houston, Paul; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Rock, Fernando L.; Sexton, Holly; Zhang, Suoming; Zhou, Yasheen; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G.; Woolhiser, Lisa; Gruppo, Veronica; Lenaerts, Anne J.; O'Malley, Theresa; Parish, Tanya; Cooper, Christopher B.; Waters, M. Gerard; Ma, Zhenkun; Ioerger, Thomas R.; Sacchettini, James C.; Rullas, Joaquín; Angulo-Barturen, Iñigo; Pérez-Herrán, Esther; Mendoza, Alfonso; Barros, David; Cusack, Stephen; Plattner, Jacob J.

    2016-01-01

    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid. PMID:27503647

  4. Statistical Evaluation of the Rodin–Ohno Hypothesis: Sense/Antisense Coding of Ancestral Class I and II Aminoacyl-tRNA Synthetases

    PubMed Central

    Chandrasekaran, Srinivas Niranj; Yardimci, Galip Gürkan; Erdogan, Ozgün; Roach, Jeffrey; Carter, Charles W.

    2013-01-01

    We tested the idea that ancestral class I and II aminoacyl-tRNA synthetases arose on opposite strands of the same gene. We assembled excerpted 94-residue Urgenes for class I tryptophanyl-tRNA synthetase (TrpRS) and class II Histidyl-tRNA synthetase (HisRS) from a diverse group of species, by identifying and catenating three blocks coding for secondary structures that position the most highly conserved, active-site residues. The codon middle-base pairing frequency was 0.35 ± 0.0002 in all-by-all sense/antisense alignments for 211 TrpRS and 207 HisRS sequences, compared with frequencies between 0.22 ± 0.0009 and 0.27 ± 0.0005 for eight different representations of the null hypothesis. Clustering algorithms demonstrate further that profiles of middle-base pairing in the synthetase antisense alignments are correlated along the sequences from one species-pair to another, whereas this is not the case for similar operations on sets representing the null hypothesis. Most probable reconstructed sequences for ancestral nodes of maximum likelihood trees show that middle-base pairing frequency increases to approximately 0.42 ± 0.002 as bacterial trees approach their roots; ancestral nodes from trees including archaeal sequences show a less pronounced increase. Thus, contemporary and reconstructed sequences all validate important bioinformatic predictions based on descent from opposite strands of the same ancestral gene. They further provide novel evidence for the hypothesis that bacteria lie closer than archaea to the origin of translation. Moreover, the inverse polarity of genetic coding, together with a priori α-helix propensities suggest that in-frame coding on opposite strands leads to similar secondary structures with opposite polarity, as observed in TrpRS and HisRS crystal structures. PMID:23576570

  5. The Inhibition of Folylpolyglutamate Synthetase (folC) in the Prevention of Drug Resistance in Mycobacterium tuberculosis by Traditional Chinese Medicine

    PubMed Central

    Hung, Tzu-Chieh; Chen, Kuen-Bao; Lee, Wen-Yuan

    2014-01-01

    Tuberculosis (TB) is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM) database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis. PMID:25050369

  6. Assignment of the human MARS gene, encoding methioninyl-tRNA synthetase, to chromosome 12 using human X Chinese hamster cell hybrids.

    PubMed

    Cirullo, R E; Wasmuth, J J

    1984-05-01

    We have isolated interspecific somatic cell hybrids between a temperature-sensitive Chinese hamster ovary (CHO) cell methioninyl -tRNA synthetase mutant and human peripheral leukocytes. The hybrids were selected at 39 degrees C which requires the retention and expression of the human gene, MARS , which complements the defective CHO gene. In vitro heat-inactivation experiments on the methioninyl -tRNA synthetase activity in cell-free extracts from heat-resistant hybrids indicate that the human form of this enzyme and, therefore, the human MARS gene is present in hybrid cells. Cytogenetic analysis of three independent temperature-resistant hybrids revealed the presence of a single human chromosome, number 12. Two other independent hybrids examined contained human chromosome 12 as well as a second human chromosome. Electrophoretic analysis of extracts from hybrid cell lines for a human chromosome 12 marker isozyme, LDH-B, showed a pattern of heterotetrameric bands consistent with the presence of the human form of this enzyme in these cells. The correlation between the presence of the human form of methioninyl -tRNA synthetase and human chromosome 12 in temperature-resistant hybrids indicates that the human MARS locus is located on this chromosome.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  13. 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

  14. 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

  15. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  16. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  17. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  18. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.)

    DOE PAGES

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; ...

    2015-08-24

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII andmore » GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Lastly, our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.« less

  4. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

    PubMed

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M

    2015-11-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

  5. Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe.

    PubMed

    Perysinakis, A; Kinghorn, J R; Drainas, C

    1995-06-01

    Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

  6. Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

    PubMed Central

    Alexandrova, Jana; Paulus, Caroline; Rudinger-Thirion, Joëlle; Jossinet, Fabrice; Frugier, Magali

    2015-01-01

    The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5′-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases. PMID:26327585

  7. Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis.

    PubMed

    Esaki, Shingo; Malkaram, Sridhar A; Zempleni, Janos

    2012-04-01

    Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

  8. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    PubMed

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  9. 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

  10. 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

  11. 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.

  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. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    SciTech Connect

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade

  4. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

    PubMed Central

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-01-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically. PMID:26836257

  5. Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase

    SciTech Connect

    Noinaj, Nicholas; Wattanasak, Rut; Lee, Duck-Yeon; Wally, Jeremy L.; Piszczek, Grzegorz; Chock, P. Boon; Stadtman, Thressa C.; Buchanan, Susan K.

    2012-03-26

    Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg{sup 2+} and K{sup +} and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS{sup C17S}) in apo form (without ATP bound). EcSPS{sup C17S} crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

  6. Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

    PubMed Central

    Garrow, T A; Admon, A; Shane, B

    1992-01-01

    A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria. PMID:1409616

  7. Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

    SciTech Connect

    Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim; Narasimhan, Lakshmi; Holler, Tod; McGrath, Teresa; Beattie, Bryan; Fauman, Eric; Yan, Chunhong; Heaslet, Holly; Walter, Richard; Finzel, Barry; Ohren, Jeffrey; McConnell, Patrick; Braden, Timothy; Sun, Fang; Spessard, Cindy; Banotai, Craig; Al-Kassim, Loola; Ma, Weijun; Wengender, Paul; Kole, Denis; Garceau, Norman; Toogood, Peter; Liu, Jia

    2008-07-08

    In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

  8. A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

    PubMed Central

    Houman, Fariba; Rho, Seung Bae; Zhang, Jiansu; Shen, Xiaoyu; Wang, Chien-Chia; Schimmel, Paul; Martinis, Susan A.

    2000-01-01

    Mitochondrial leucyl-tRNA synthetase (LeuRS) in the yeast Saccharomyces cerevisiae provides two essential functions. In addition to aminoacylation, LeuRS functions in RNA splicing. The details of how it came to act in splicing are not known. Here we show that Mycobacterium tuberculosis and human mitochondrial LeuRSs can substitute in splicing for the S. cerevisiae mitochondrial LeuRS. Mutations of yeast mitochondrial LeuRS that had previously been shown to abolish splicing activity also eliminate splicing by the M. tuberculosis enzyme. These results suggest the role of LeuRS in splicing in yeast mitochondria results from features of the enzyme that are broadly conserved in evolution. These features are not likely to be designed for splicing per se, but instead have been adopted in yeast for that purpose. PMID:11087829

  9. Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation.

    PubMed

    Masalkar, Pintu D; Roberts, Daniel M

    2015-01-16

    Legume root nodule glutamine synthetase (GS) catalyzes the assimilation of ammonia produced by nitrogen fixation. Two GS isoform subtypes (GS1β and GS1γ) are present in soybean nodules. GS1γ isoforms differ from GS1β isoforms in terms of their susceptibility to reversible inhibition by intersubunit disulfide bond formation between C159 and C92 at the shared active site at subunit interfaces. Although nodule GS enzymes share 86% amino acid sequence identity, analytical ultracentrifugation experiments showed that GS1γ is a dodecamer, whereas the GS1β is a decamer. It is proposed that this difference contributes to the differential thiol sensitivity of each isoform, and that GS1γ1 may be a target of thiol-based regulation.

  10. IL-1beta stimulates argininosuccinate synthetase gene expression through NF-kappaB in Caco-2 cells.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Husson, Annie

    2005-05-01

    Argininosuccinate synthetase (ASS) is limiting the arginine synthesis and can be stimulated by immunostimulants. We previously identified a putative NF-kappaB element in the human ASS gene promoter but its functionality was unknown (Husson et al., Eur. J. Biochem. 270 (2003) 1887). In the present study, using Caco-2 cells, a human enterocyte line, we demonstrate that IL-1beta rapidly induces the expression of the ASS gene at a transcriptional level through NF-kappaB activation. Using gel shift assay and double-strand oligonucleotide sequence of the identified putative NF-kappaB binding site of the ASS promoter, we provide evidence that NF-kappaB may functionally interact with this element.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

    PubMed

    Hauf, Ksenia; Kayumov, Airat; Gloge, Felix; Forchhammer, Karl

    2016-02-12

    TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

  17. Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling*

    PubMed Central

    Leroux, Alejandro E.; Haanstra, Jurgen R.; Bakker, Barbara M.; Krauth-Siegel, R. Luise

    2013-01-01

    In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)2)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as Ki values for GSH and T(SH)2 of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a Km value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated. PMID:23814051

  18. N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1

    SciTech Connect

    Liu Honglin; Peng, Xiaohui; Zhao Fang; Zhang Guobin; Tao Ye; Luo Zhaofeng; Li Yang; Teng Maikun; Li Xu Wei Shiqiang

    2009-02-20

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of {alpha}-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO{sub 4}{sup 3-} allosteric site where PO{sub 4}{sup 3-} functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

  19. A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects*

    PubMed Central

    Wang, Yong; Zhou, Xiao-Long; Ruan, Zhi-Rong; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2016-01-01

    Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs. PMID:26811336

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-11-01

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

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

    PubMed

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

    2008-10-15

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

  8. 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.

  9. 2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

    PubMed Central

    Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

    2015-01-01

    2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

  10. Role of 4-Hydroxybutyrate-CoA Synthetase in the CO2 Fixation Cycle in Thermoacidophilic Archaea

    SciTech Connect

    Hawkins, AS; Han, YJ; Bennett, RK; Adams, MWW; Kelly, RM

    2013-02-08

    Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific genes, except for the one responsible for ligation of coenzyme A (CoA) to 4HB. Although several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. In this report, transcriptomic analysis of cells grown under strict H-2-CO2 autotrophy was consistent with the involvement of Msed_0406 and Msed_0394. Recombinant versions of these enzymes catalyzed the ligation of CoA to 4HB, with similar affinities for 4HB (K-m values of 1.9 and 1.5 mM for Msed_0406 and Msed_0394, respectively) but with different rates (1.69 and 0.22 mu mol x min(-1) x mg(-1) for Msed_0406 and Msed_0394, respectively). Neither Msed_0406 nor Msed_0394 have close homologs in other Sulfolobales, although low sequence similarity is not unusual for acyl-adenylate-forming enzymes. The capacity of these two enzymes to use 4HB as a substrate may have arisen from simple modifications to acyl-adenylate-forming enzymes. For example, a single amino acid substitution (W424G) in the active site of the acetate/propionate synthetase (Msed_1353), an enzyme that is highly conserved among the Sulfolobales, changed its substrate specificity to include 4HB. The identification of the 4-HB CoA synthetase now completes the set of enzymes comprising the 3HP/4HB cycle.

  11. Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate.

    PubMed

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

    2011-03-01

    Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions of people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg(2+) ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of Escherichia coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA(Met) complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.

  12. Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

    PubMed Central

    Murali, N; Lin, Y; Mechulam, Y; Plateau, P; Rao, B D

    1997-01-01

    The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain. Images FIGURE 6 PMID:9129831

  13. Response to nitrate/ammonium nutrition of tomato (Solanum lycopersicum L.) plants overexpressing a prokaryotic NH4(+)-dependent asparagine synthetase.

    PubMed

    Martínez-Andújar, Cristina; Ghanem, Michel Edmond; Albacete, Alfonso; Pérez-Alfocea, Francisco

    2013-05-01

    Nitrogen availability is an important limiting factor for plant growth. Although NH4(+) assimilation is energetically more favorable than NO3(-), it is usually toxic for plants. In order to study if an improved ammonium assimilatory metabolism could increase the plant tolerance to ammonium nutrition, tomato (Solanum lycopersicum L. cv P-73) plants were transformed with an NH4(+)-dependent asparagine synthetase (AS-A) gene from Escherichia coli (asnA) under the control of a PCpea promoter (pea isolated constitutive promotor). Homozygous (Hom), azygous (Az) asnA and wild type (WT) plants were grown hydroponically for 6 weeks with normal Hoagland nutrition (NO3(-)/NH4(+)=6/0.5) and high ammonium nutrition (NO3(-)/NH4(+)=3.5/3). Under Hoagland's conditions, Hom plants produced 40-50% less biomass than WT and Az plants. However, under NO3(-)/NH4(+)=3.5/3 the biomass of Hom was not affected while it was reduced by 40-70% in WT and Az plants compared to Hoagland, respectively. The Hom plants accumulated 1.5-4 times more asparagine, glycine, serine and soluble proteins and registered higher glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in the light-adapted leaves than the other genotypes, but had similar NH4(+) and NO3(-) levels in all conditions. In the dark-adapted leaves, a protein catabolism occurred in the Hom plants with a concomitant 25-40% increase in organic acid concentration, while asparagine accumulation registered the highest values. The aforementioned processes might be responsible for a positive energetic balance as regards the futile cycle of the transgenic protein synthesis and catabolism. This explains growth penalty under standard nutrition and growth stability under NO3(-)/NH4(+)=3.5/3, respectively.

  14. Calpain Cleaves Most Components in the Multiple Aminoacyl-tRNA Synthetase Complex and Affects Their Functions.

    PubMed

    Lei, Hui-Yan; Zhou, Xiao-Long; Ruan, Zhi-Rong; Sun, Wei-Cheng; Eriani, Gilbert; Wang, En-Duo

    2015-10-23

    Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNA(Gln) and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca(2+) and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC.

  15. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains

    PubMed Central

    Soupene, Eric; Kuypers, Frans A

    2006-01-01

    Background The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL) genes have been identified with as many as 3 different transcript variants for each. Results Acyl-CoA Synthetase Long-chain member 6 (ACSL6) is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr). Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs. Conclusion We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes. PMID:16834775

  16. 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.

  17. 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

  18. 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

  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. 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...

  3. 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.

  4. 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.

  5. 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

  6. 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

  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. 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.

  9. 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

  10. 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.

  11. 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

  12. A case of anti-aminoacyl tRNA synthetase antibody syndrome complicated by hemophagocytic syndrome.

    PubMed

    Azuma, Kota; Tamura, Masao; Kurajoh, Masafumi; Hosono, Yuji; Nakajima, Ran; Tsuboi, Kazuyuki; Abe, Takeo; Ogita, Chie; Yokoyama, Yuichi; Furukawa, Tetsuya; Yoshikawa, Takahiro; Saito, Atsushi; Nishioka, Aki; Sekiguchi, Masahiro; Azuma, Naoto; Kitano, Masayasu; Tsunoda, Shinichiro; Omura, Koichiro; Koyama, Hidenori; Matsui, Kiyoshi; Mimori, Tsuneyo; Sano, Hajime

    2016-01-01

      A 48-year-old woman had suffered from a fever and general fatigue, and visited the other hospital for fever elevation in November 2013, at which time interstitial lung disease was revealed. In January 2014, she experienced an eruption in the hand and developed peripheral blood flow damage. Under a diagnosis of adult Still's disease, the patient was administered 0.5 mg of betamethasone as well as cyclosporin at 75 mg/day. In November 2014, general fatigue, fever, and headache were noted, while MRI revealed an enlarged hypophysis and laboratory findings were positive for the anti-pituitary cell antibody, thus a diagnosis of autoimmune hypophysitis was made. Although disease activity was low, she requested hospitalization and was admitted by the Division of Endocrinology and Metabolism at our hospital in May 2015, though only observed. Fever developed again, along with interstitial lung disease, Raynaud's phenomenon, and pain in the crural area again, and we considered the possibility of another disease. After stopping administration of betamethasone and cyclosporin, we made a diagnosis of anti-aminoacyl tRNA synthetase antibody syndrome, and administered methylprednisolone at 500 mg for 3 days as well as prednisolone at 35 mg/day following steroid pulse therapy. Although her condition soon improved, fever, muscle pain, and pancytopenia returned after 3 days. Bone marrow findings revealed the existence of hemophagocytosis, for which we again gave methylprednisolone at 500 mg for 3 days and cyclosporin at 125 mg/day. Thereafter, the patient recovered and was discharged from the hospital.

  13. Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

    PubMed

    Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

    2016-06-21

    Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

  14. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

    SciTech Connect

    Suzuki, Y.; Aoki, Y.; Ishida, Y.

    1994-09-01

    Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.

  15. Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Luo, Yi-Pey; Chen, Yi-Ling

    2015-01-01

    Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin‑embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer.

  16. Promoter demethylation of cystathionine-β-synthetase gene contributes to inflammatory pain in rats.

    PubMed

    Qi, Feihu; Zhou, Youlang; Xiao, Ying; Tao, Jin; Gu, Jianguo; Jiang, Xinghong; Xu, Guang-Yin

    2013-01-01

    Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

  17. Selenophosphate Synthetase 1 is an Essential Protein with Roles in Regulation of Redox Homeostasis in Mammals

    PubMed Central

    Tobe, Ryuta; Carlson, Bradley A.; Huh, Jang Hoe; Castro, Nadia P.; Xu, Xue-Ming; Tsuji, Petra A.; Lee, Sang-Goo; Bang, Jeyoung; Na, Ji-Woon; Kong, Young-Yun; Beaglehole, Daniel; Southon, Eileen; Seifried, Harold; Tessarollo, Lino; Salomon, David S.; Schweizer, Ulrich; Gladyshev, Vadim N.; Hatfield, Dolph L.; Lee, Byeong Jae

    2016-01-01

    Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogs, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyzes the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1 knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by E8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development, and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione-S-transferase omega 1. To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Further, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homeostasis and controlling cell growth. PMID:27208177

  18. Mammalian folylpoly-. gamma. -glutamate synthetase. 2. Substrate specificity and kinetic properties

    SciTech Connect

    Cichowicz, D.J.; Shane, B.

    1987-01-27

    The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and L-(/sup 14/C)glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis while 5- and 10-position substitutions of the folate molecule impair catalysis. k/sub cat/ values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The K/sub m/ for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and ..beta..,..gamma..-methylene-ATP, ..beta..,..gamma..-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P/sup 1/,P/sup 5/-di(adenosine-5') pentaphosphate, and free ATP/sup 4 -/ are potent inhibitors of the reaction.

  19. The central loop of Escherichia coli glutamine synthetase is flexible and functionally passive.

    PubMed

    Pearson, Josh T; Dabrowski, Michael J; Kung, Irene; Atkins, William M

    2005-04-15

    Bacterial glutamine synthetases (GSs) are dodecameric aggregates comprised of two face-to-face hexameric rings, which form a cylindrical aqueous channel. Available crystal structures indicate that each subunit provides a 'central loop' that protrudes into this channel. Residues on either side of this loop contribute directly to substrate or metal ion cofactor binding. Although it has been suggested that this conspicuous structural feature may be functionally important, a systematic structure-function analysis of this loop has not been done. Here, we examine the behavior of a cysteine mutant, E165C, which yields inter-subunit disulfide bonds connecting the central loops. The inter-subunit disulfide bonds are readily detected by electrospray ionization mass spectrometry. Based on molecular models, the disulfide bonds would form only if the engineered cysteines on adjacent subunits moved approximately 5 A. Surprisingly, inter-subunit disulfide bonds between the central loops caused no detectable changes in the KMs for glutamate or ATP, nor the KD for either ATP or the transition state analog (L)-methionine sulfoximine (MSOX). Furthermore, covalent and quantitative adduction of the E165C mutant with iodo-acetamido-pyrene yielded nearly fully active enzyme bearing fluorescent pyrene excimers. The relative contribution of pyrene monomers to excimers in the steady state fluorescence is temperature dependent, suggesting thermal equilibrium between loop conformational states. However, the monomer-excimer ratio is independent of ligands such as MSOX, glutamate, or Mn2+. These results validate the suspected flexibility of the central loop, but raise significant doubt about its direct functional role in GS catalysis via conformational switching, including the proposed regulation of GS via ADP-ribosylation within this loop.

  20. Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides

    PubMed Central

    Davies, Richard C.; Neuberger, Albert

    1973-01-01

    1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted α-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed. PMID:4542566