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Sample records for human lanthionine synthetase

  1. Mechanistic Studies of Ser/Thr Dehydration Catalyzed by a Member of the LanL Lanthionine Synthetase Family†

    Science.gov (United States)

    2011-01-01

    Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model. PMID:21229987

  2. Mechanistic studies of Ser/Thr dehydration catalyzed by a member of the LanL lanthionine synthetase family.

    Science.gov (United States)

    Goto, Yuki; Okesli, Ayşe; van der Donk, Wilfred A

    2011-02-08

    Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model.

  3. Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2.

    Directory of Open Access Journals (Sweden)

    Pinyi Lu

    Full Text Available Lanthionine synthetase component C-like protein 2 (LANCL2 is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA, a plant hormone with anti-diabetic and anti-inflammatory effects.The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1 as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610 in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses.LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.

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

    Science.gov (United States)

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

    2010-01-21

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bonnefond, Luc; Frugier, Magali; Touzé, Elodie; Lorber, Bernard; Florentz, Catherine; Giegé, Richard, E-mail: r.giege@ibmc.u-strasbg.fr; Rudinger-Thirion, Joëlle; Sauter, Claude [Département ‘Machineries Traductionnelles’, Architecture et Réactivité de l’ARN, Université Louis Pasteur de Strasbourg, CNRS, IBMC, 15 Rue René Descartes, 67084 Strasbourg (France)

    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.

  6. Biocytin synthetase activity in human milk as assessed by high-performance liquid chromatography.

    Science.gov (United States)

    Oizumi, J; Hayakawa, K

    1993-01-29

    A reversed-phase liquid chromatographic assay for biocytin synthetase activity has been developed. By this method, biocytin synthetase, isolated to homogeneity from human milk, was found to synthetize biocytin from biotin and L-lysine in the presence of ATP and magnesium ion(s). Both ATP and magnesium ion(s) were required for the synthesis of biocytin. Equal molar amounts of ADP and ATP were produced and consumed, respectively, in the course of the production of the same molar amount of biocytin; however, production of AMP was not observed. Biocytin synthetase Michaelis constants were 2.5, 1.8, and 0.11 mM for biotin, L-lysine, and ATP, respectively. Biocytin synthetase from milk was shown to synthesize biocytin in a stoichiometric amount.

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

    Energy Technology Data Exchange (ETDEWEB)

    Webb, G.C.; Vaska, V.L.; Ford, J.H. [Queen Elizabeth Hospital, Woodville (Australia)] [and others

    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.

  8. Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine

    DEFF Research Database (Denmark)

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman

    2004-01-01

    . Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease...... or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl...

  9. Expression of human aspartyl-tRNA synthetase in COS cells.

    Science.gov (United States)

    Escalante, C; Qasba, P K; Yang, D C

    1994-11-09

    Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.

  10. Structural context for mobilization of a human tRNA synthetase from its cytoplasmic complex.

    Science.gov (United States)

    Fang, Pengfei; Zhang, Hui-Min; Shapiro, Ryan; Marshall, Alan G; Schimmel, Paul; Yang, Xiang-Lei; Guo, Min

    2011-05-17

    Human lysyl-tRNA synthetase is bound to the multi-tRNA synthetase complex (MSC) that maintains and regulates the aminoacylation and nuclear functions of LysRS. The p38 scaffold protein binds LysRS to the MSC and, only with the appropriate cue, mobilizes LysRS for redirection to the nucleus to interact with the microphthalmia associated transcription factor (MITF). In recent work, an (α(2))(2) LysRS tetramer crystallized to yield a high-resolution structure and raised the question of how LysRS is arranged (dimer or tetramer) in the MSC to interact with p38. To understand the structural organization of the LysRS-p38 complex that regulates LysRS mobilization, we investigated the complex by use of small angle X-ray scattering and hydrogen-deuterium exchange with mass spectrometry in solution. The structure revealed a surprising α(2)β(1):β(1)α(2) organization in which a dimeric p38 scaffold holds two LysRS α(2) dimers in a parallel configuration. Each of the N-terminal 48 residues of p38 binds one LysRS dimer and, in so doing, brings two copies of the LysRS dimer into the MSC. The results suggest that this unique geometry, which reconfigures the LysRS tetramer from α(2):α(2) to α(2)β(1):β(1)α(2), is designed to control both retention and mobilization of LysRS from the MSC.

  11. Insights into substrate promiscuity of human seryl-tRNA synthetase.

    Science.gov (United States)

    Holman, Kaitlyn M; Puppala, Anupama K; Lee, Jonathan W; Lee, Hyun; Simonović, Miljan

    2017-11-01

    Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNA Ser ) and the noncognate selenocysteine tRNA (tRNA Sec ). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNA Ser , tRNA Sec , and 10 mutant and chimeric constructs in which elements of tRNA Ser were transposed onto tRNA Sec We show that human SerRS only subtly prefers tRNA Ser to tRNA Sec , and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNA Sec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNA Sec and tRNA Ser dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNA Sec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis. © 2017 Holman et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

    Directory of Open Access Journals (Sweden)

    Manuel Castro de Moura

    2011-11-01

    Full Text Available 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.

  13. Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

    Science.gov (United States)

    Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-01-01

    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. PMID:22140588

  14. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    Science.gov (United States)

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  15. Oligoadenylate synthetase 1 (OAS1 expression in human breast and prostate cancer cases, and its regulation by sex steroid hormones

    Directory of Open Access Journals (Sweden)

    Cláudio Jorge Maia

    2016-06-01

    Full Text Available Oligoadenylate synthetase 1 (OAS1 is an interferon-induced protein characterised by its capacity to catalyse the synthesis of 2ʹ-5ʹ-linked oligomers of adenosine from adenosine triphosphate (2-5A. The 2-5A binds to a latent Ribonuclease L (RNase L, which subsequently dimerises into its active form and may play an important role in the control of cell growth, differentiation and apoptosis. Previously, our research group identified OAS1 as a differentially-expressed gene in breast and prostate cancer cell lines when compared to normal cells. This study evaluates: i the expression of OAS1 in human breast and prostate cancer specimens; and ii the effect of sex steroid hormones in regulating the expression of OAS1 in breast (MCF-7 and prostate (LNCaP cancer cell lines. The obtained results showed that OAS1 expression was down-regulated in human infiltrative ductal carcinoma of breast, adenocarcinoma of prostate, and benign prostate hyperplasia, both at mRNA and protein level. In addition, OAS1 expression was negatively correlated with the progression of breast and prostate cancer. With regards to the regulation of OAS1 gene, it was demonstrated that 17β-estradiol (E2 down-regulates OAS1 gene in MCF-7 cell lines, an effect that seems to be dependent on the activation of oestrogen receptor (ER. On the other hand, 5α-dihydrotestosterone (DHT treatment showed no effect on the expression of OAS1 in LNCaP cell lines. The lower levels of OAS1 in breast and prostate cancer cases indicated that the OAS1/RNaseL apoptotic pathway may be compromised in breast and prostate tumours. Moreover, the present findings suggested that this effect may be enhanced by oestrogen in ER-positive breast cancers.

  16. Interferon-mediated antiviral state in human MRC5 cells in the absence of detectable levels of 2-5A synthetase and protein kinase.

    Science.gov (United States)

    Meurs, E; Hovanessian, A G; Montagnier, L

    1981-02-01

    Treatment of human HeLa and MRC5 cells with human alpha (leukocyte) and beta (fibroblast) interferon results in the development of an antiviral state against two types of viruses: vesicular stomatitis virus (rhabdovirus) and encephalomyocarditis virus (picornavirus). These cells, however, differ in their ability to synthesize the two double-stranded (ds) RNA-dependent enzymatic activities, pppA(2'p5'A)n synthetase (2-5A synthetase) and protein kinase which have been reported to be induced in several cell lines by interferon. Both the 2-5A synthetase and the protein kinase are enhanced by several fold in HeLa cells on treatment with interferon. In contrast, neither the 2-5A synthetase nor the protein kinase can be detected in MRC5 cell treated or not treated with interferon. The lack of detection of the 2-5A synthetase in MRC5 cells is not associated with the absence of the other components of the 2-5A system (2-5A dependent nuclease and 2'-phosphodiesterase). We have previously shown that MRC5 cells are sensitive to the action of 2-5A and furthermore the inhibitory action of 2-5A on these cells is transient. Mixing experiments between HeLa and MRC5 cell fractions after partial purification on columns of poly(I).poly(C)-Sepharose, showed that the absence of detection of the protein kinase activity in MRC5 cells cannot be attributed to the presence of phosphatases or other inhibitors of phosphorylation in control or interferon-treated MRC5 cell extracts. In addition, we show that the interferon-mediated protein kinase activity in HeLa cell extracts can be precipitated by treatment at pH 5, a procedure which leads to an enhanced level of detectable protein kinase activity in general. Once again, however, MRC5 cell extracts fail to show any interferon-mediated protein kinase activity. These results suggest that either the two enzyme activities are not necessary for the development of the antiviral response induced by interferon or the intracellular events leading to

  17. USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME'S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE

    OpenAIRE

    DÍEZ FERNÁNDEZ, CARMEN

    2016-01-01

    [EN] Carbamoyl phosphate synthetase 1 (CPS1), a 1462-residue mitochondrial enzyme, catalyzes the entry of ammonia into the urea cycle, which converts ammonia, the neurotoxic waste product of protein catabolism, into barely toxic urea. The urea cycle inborn error and rare disease CPS1 deficiency (CPS1D) is inherited with mendelian autosomal recessive inheritance, being due to CPS1 gene mutations (>200 mutations reported), and causing life-threatening hyperammonemia. We have produced recomb...

  18. Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine.

    Science.gov (United States)

    Liu, Jianming; Yang, Xiang-Lei; Ewalt, Karla L; Schimmel, Paul

    2002-12-03

    Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.

  19. Cytosolic glutamine synthetase

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie; Eriksson, Ulf Dennis; Møller, Inge Skrumsager

    2014-01-01

    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...

  20. MiSynPat: An integrated knowledge base linking clinical, genetic, and structural data for disease-causing mutations in human mitochondrial aminoacyl-tRNA synthetases.

    Science.gov (United States)

    Moulinier, Luc; Ripp, Raymond; Castillo, Gaston; Poch, Olivier; Sissler, Marie

    2017-10-01

    Numerous mutations in each of the mitochondrial aminoacyl-tRNA synthetases (aaRSs) have been implicated in human diseases. The mutations are autosomal and recessive and lead mainly to neurological disorders, although with pleiotropic effects. The processes and interactions that drive the etiology of the disorders associated with mitochondrial aaRSs (mt-aaRSs) are far from understood. The complexity of the clinical, genetic, and structural data requires concerted, interdisciplinary efforts to understand the molecular biology of these disorders. Toward this goal, we designed MiSynPat, a comprehensive knowledge base together with an ergonomic Web server designed to organize and access all pertinent information (sequences, multiple sequence alignments, structures, disease descriptions, mutation characteristics, original literature) on the disease-linked human mt-aaRSs. With MiSynPat, a user can also evaluate the impact of a possible mutation on sequence-conservation-structure in order to foster the links between basic and clinical researchers and to facilitate future diagnosis. The proposed integrated view, coupled with research on disease-related mt-aaRSs, will help to reveal new functions for these enzymes and to open new vistas in the molecular biology of the cell. The purpose of MiSynPat, freely available at http://misynpat.org, is to constitute a reference and a converging resource for scientists and clinicians. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  1. Mutations in the mitochondrial methionyl-tRNA synthetase cause a neurodegenerative phenotype in flies and a recessive ataxia (ARSAL in humans.

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    Vafa Bayat

    Full Text Available An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS, and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

  2. Association of human mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol does not require other viral proteins

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    Lydia Kobbi

    2016-06-01

    Full Text Available In human, the cytoplasmic (cLysRS and mitochondrial (mLysRS species of lysyl-tRNA synthetase are encoded by a single gene. Following HIV-1 infection, mLysRS is selectively taken up into viral particles along with the three tRNALys isoacceptors. The GagPol polyprotein precursor is involved in this process. With the aim to reconstitute in vitro the HIV-1 tRNA3Lys packaging complex, we first searched for the putative involvement of another viral protein in the selective viral hijacking of mLysRS only. After screening all the viral proteins, we observed that Vpr and Rev have the potential to interact with mLysRS, but that this association does not take place at the level of the assembly of mLysRS into the packaging complex. We also show that tRNA3Lys can form a ternary complex with the two purified proteins mLysRS and the Pol domain of GagPol, which mimicks its packaging complex.

  3. Degenerative knee joint disease in mice lacking 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (Papss2) activity: a putative model of human PAPSS2 deficiency-associated arthrosis.

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    Ford-Hutchinson, Alice F; Ali, Zenobia; Seerattan, Ruth A; Cooper, David M L; Hallgrímsson, Benedikt; Salo, Paul T; Jirik, Frank R

    2005-05-01

    Murine brachymorphism (bm) results from an autosomal recessive mutation of the Papss2 gene that encodes 3'-phosphoadenosine 5'-phosphosulfate synthetase 2, one of the principal enzymes required for the sulfation of extracellular matrix molecules in cartilage and other tissues. A spondyloepimetaphyseal dysplasia has been identified in Pakistani kindred having a mutation of PAPSS2. In addition to skeletal malformations that include short stature evident at birth due to limb shortening, brachydactyly, and kyphoscoliosis, affected individuals demonstrate premature onset degenerative joint disease. We investigated whether loss of Papss2 activity would similarly lead to degenerative joint disease in mice. Mice carrying the bm mutation on a C57BL/6 background were obtained from the Jackson Laboratory. Limbs were analyzed by micro-computed tomography (microCT) and histology. At 12 months of age both male and female bm mice exhibited severe degenerative knee joint disease, with cartilage damage being primarily evident in the patello-femoral and medial compartments. Control 12-14-month-old C57BL/6 mice, in contrast, only occasionally demonstrated minimal cartilage damage. muCT imaging of bm limbs revealed shortened diaphyses associated with flared metaphyses in the proximal elements of both fore and hind limbs. Additionally, the bm hind limbs demonstrated extensive structural alterations, characterized by distortion of the patello-femoral groove, and prominent bowing of both tibia and fibula. The bm mutant, which develops severe articular cartilage lesions of the knee joint by approximately 12 months of age, represents a novel example of murine degenerative joint disease, possibly representing a model of human PAPSS2 deficiency-associated arthrosis.

  4. Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

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    Bruzzone, Santina; Basile, Giovanna; Mannino, Elena; Sturla, Laura; Magnone, Mirko; Grozio, Alessia; Salis, Annalisa; Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; De Flora, Antonio; Tossi, Vanesa; Cassia, Raul; Lamattina, Lorenzo; Zocchi, Elena

    2012-06-01

    UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation. Copyright © 2011 Wiley Periodicals, Inc.

  5. Elucidation of the structure of SA-FF22, a lanthionine-containing antibacterial peptide produced by Streptococcus pyogenes strain FF22.

    Science.gov (United States)

    Jack, R W; Carne, A; Metzger, J; Stefanović, S; Sahl, H G; Jung, G; Tagg, J

    1994-03-01

    The antibacterial peptide SA-FF22, produced by the pathogen Streptococcus pyogenes strain FF22 was purified and features of its primary and secondary structure were characterised. Mass spectrometry demonstrated the pure peptide had a mass of 2794Da while, amino acid analysis revealed the presence of the unusual, thioether amino acids lanthionine and 3-methyllanthionine; thus SA-FF22 is a member of the group of antibacterial polypeptides termed lantibiotics. Furthermore, amino acid sequencing showed a unique sequence which was blocked at position 23 by a residue of the unsaturated amino acid 2,3-didehydrobutyrine. Carboxypeptidase-Y digestion could be used to demonstrate that serine occupies the C-terminal position only after complete oxidation of the thioether amino acid bridges, suggesting that the three-dimensional structure of the native peptide may prevent access of the enzyme to the C-terminus. Fragmentation of the native peptide with a variety of proteolytic enzymes failed to yield a peptide containing less than all three of the cross-linked lanthionine and methyllanthionine residues and demonstrated that all three thioether bridges overlapped. Analysis of the circular dichroism of SA-FF22 in various concentrations of 2,2,2-trifluoroethanol in water, SDS micelles and in the presence of artificial phospholipid vesicles suggested that there is significant change in its secondary structure from aqueous to lipophilic environments.

  6. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

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

    NARCIS (Netherlands)

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

    1990-01-01

    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

  8. Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

    DEFF Research Database (Denmark)

    Durkin, M E; Jäger, A C; Khurana, T S

    1999-01-01

    The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome...... 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we...... found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein...

  9. Genetics Home Reference: glutathione synthetase deficiency

    Science.gov (United States)

    ... with severe glutathione synthetase deficiency also develop recurrent bacterial infections. Related Information What does it mean if a disorder seems to run in my family? What is the prognosis of a genetic condition? Genetic and Rare Diseases Information Center Frequency ...

  10. Genetics Home Reference: phosphoribosylpyrophosphate synthetase superactivity

    Science.gov (United States)

    ... and PRPP also play a key role in recycling purines from the breakdown of DNA and RNA, ... of Arthritis and Musculoskeletal and Skin Diseases: Gout Educational Resources (4 links) Disease InfoSearch: Phosphoribosylpyrophosphate synthetase superactivity ...

  11. Glutathione synthetase deficiency associated with antenatal cerebral bleeding

    NARCIS (Netherlands)

    Brüggemann, L. W.; Groenendaal, F.; Ristoff, E.; Larsson, A.; Duran, M.; van Lier, J. A. C.; Dorland, L.; Berger, R.; de Koning, T. J.

    2004-01-01

    We present a newborn with glutathione synthetase deficiency and intracranial haemorrhages. Because the latter are rare in term newborns a possible relationship with glutathione synthetase deficiency will be discussed

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

    Energy Technology Data Exchange (ETDEWEB)

    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., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    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.

  13. Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

    DEFF Research Database (Denmark)

    Hartmann, R; Norby, P L; Martensen, P M

    1998-01-01

    A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range....... Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro......-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel...

  14. Growth factors regulate glutamine synthetase activity in ...

    African Journals Online (AJOL)

    Khaled

    2012-07-10

    Jul 10, 2012 ... medium; NB, nutrient broth medium; NF, nitrogen fixation medium; SDS-PAGE, sodium dodecyl sulfate ... compounds, such as amino acids, as their sole source of nitrogen. In each case, substitution of ammonia by .... polymyxa producing glutamine synthetase. Different protein patterns of the total cellular ...

  15. Retinal Vasculitis in Anti-Synthetase Syndrome.

    Science.gov (United States)

    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.]. Copyright 2016, SLACK Incorporated.

  16. Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

    Directory of Open Access Journals (Sweden)

    Diego A Alonzo

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-08-14

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

  18. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  19. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    Science.gov (United States)

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  20. Spinach Leaf Acetyl-Coenzyme a Synthetase: Purification and Characterization

    National Research Council Canada - National Science Library

    Carolyn A. Zeiher; Douglas D. Randall

    1991-01-01

    Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography...

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

    Science.gov (United States)

    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

  2. A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

    Directory of Open Access Journals (Sweden)

    Ashley L Waldron

    Full Text Available Histidyl tRNA Synthetase (HARS is a member of the aminoacyl tRNA synthetase (ARS family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

  3. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    1987-01-01

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

  5. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  6. Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1983-01-01

    A mutant of Escherichia coli with a partially defective phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase) has been characterized genetically. The genetic lesion causing the altered phosphoribosylpyrophosphate synthetase, prs, was mapped at 26 min on the linkage map by con...

  7. Evolutionary divergence of chloroplast FAD synthetase proteins

    Directory of Open Access Journals (Sweden)

    Arilla-Luna Sonia

    2010-10-01

    Full Text Available Abstract Background Flavin adenine dinucleotide synthetases (FADSs - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity.

  8. Evolutionary divergence of chloroplast FAD synthetase proteins

    Science.gov (United States)

    2010-01-01

    Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity. PMID:20955574

  9. The glutamine synthetase gene family in Populus

    Directory of Open Access Journals (Sweden)

    Cánovas Francisco M

    2011-08-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

  11. NCBI nr-aa BLAST: CBRC-AGAM-07-0029 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0029 ref|ZP_01423834.1| Lanthionine synthetase C-like [Herpetosiphon aura...ntiacus ATCC 23779] gb|EAU19386.1| Lanthionine synthetase C-like [Herpetosiphon aurantiacus ATCC 23779] ZP_01423834.1 2e-20 25% ...

  12. Inactivation of Escherichia coli phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP. Identification of active site lysines

    DEFF Research Database (Denmark)

    Hilden, Ida; Hove-Jensen, Bjarne; Harlow, Kenneth W.

    1995-01-01

    . Cysteine 229 may also be labeled by oATP. Of these four residues, Lys193 is completely conserved within the family of PRPP synthetases, and Lys181 is found at a position in the sequence where the cognate amino acid (Asp181) in human isozyme I PRPP synthetase has been previously implicated in the regulation...... of enzymatic activity. These results imply a functional role for at least two of the identified amino acid residues.......M. Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit. oATP served as a substrate in the presence of ribose-5...

  13. Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.

    Directory of Open Access Journals (Sweden)

    Inês Loureiro

    Full Text Available Asparagine synthetase (AS catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A, known as strictly ammonia-dependent, and asparagine synthetase B (AS-B, which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A and Trypanosoma brucei (TbAS-A: the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

  14. Glutamine synthetase is essential in early mouse embryogenesis

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Vermeulen, Jacqueline L. M.; Lamers, Wouter H.; van Roon, Maria A.

    2007-01-01

    Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role

  15. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    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.

  16. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  17. Restoration Of Glutamine Synthetase Activity, Nitric Oxide Levels ...

    African Journals Online (AJOL)

    Background: Propolis has been proposed to be protective on neurodegenerative disorders. To understand the neuroprotective effects of honeybee propolis, glutamine synthetase (GS) activity, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and total antioxidant status (TAS) were studied in different brain ...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    1972-01-01

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

  20. Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess.

    Science.gov (United States)

    Huat, Lim Boon; Garcia, Alfonso Olivos; Ning, Tan Zi; Kin, Wong Weng; Noordin, Rahmah; Azham, Siti Shafiqah Anaqi; Jie, Lee Zhi; Ching, Guee Cher; Chong, Foo Phiaw; Dam, Pim Chau

    2014-06-01

    To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

  1. Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess

    Science.gov (United States)

    Huat, Lim Boon; Garcia, Alfonso Olivos; Ning, Tan Zi; Kin, Wong Weng; Noordin, Rahmah; Azham, Siti Shafiqah Anaqi; Jie, Lee Zhi; Ching, Guee Cher; Chong, Foo Phiaw; Dam, Pim Chau

    2014-01-01

    Objective To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Methods Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. Results A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. Conclusions This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations. PMID:25182945

  2. Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency.

    Science.gov (United States)

    Ben Ameur, Salma; Aloulou, Hajer; Nasrallah, Fehmi; Kamoun, Thouraya; Kaabachi, Naziha; Hachicha, Mongia

    2015-02-01

    Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

  3. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  4. Aminoacyl-tRNA Synthetase Complexes in Evolution

    Directory of Open Access Journals (Sweden)

    Svitlana Havrylenko

    2015-03-01

    Full Text Available 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.

  5. A stochastic modeling of isotope exchange reactions in glutamine synthetase

    Science.gov (United States)

    Kazmiruk, N. V.; Boronovskiy, S. E.; Nartsissov, Ya R.

    2017-11-01

    The model presented in this work allows simulation of isotopic exchange reactions at chemical equilibrium catalyzed by a glutamine synthetase. To simulate the functioning of the enzyme the algorithm based on the stochastic approach was applied. The dependence of exchange rates for 14C and 32P on metabolite concentration was estimated. The simulation results confirmed the hypothesis of the ascertained validity for preferred order random binding mechanism. Corresponding values of K0.5 were also obtained.

  6. Crystal structure of carbapenam synthetase (CarA).

    Science.gov (United States)

    Miller, Matthew T; Gerratana, Barbara; Stapon, Anthony; Townsend, Craig A; Rosenzweig, Amy C

    2003-10-17

    Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.

  7. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

    Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

  8. ß-Lysine discrimination by lysyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Gilreath, Marla S; Roy, Hervé; Bullwinkle, Tammy J

    2011-01-01

    Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate a- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site gui...

  9. Fatty Acid Synthetase of Spinacia oleracea Leaves 1

    Science.gov (United States)

    Shimakata, Takashi; Stumpf, Paul K.

    1982-01-01

    The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthetase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli. From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system. PMID:16662382

  10. The eucaryotic aminoacyl-tRNA synthetase complex: suggestions for its structure and function

    Science.gov (United States)

    1984-01-01

    Aminoacyl-tRNA synthetases from eucaryotic cells generally are isolated as high molecular weight complexes comprised of multiple synthetase activities, and often containing other components as well. A model is proposed for the synthetase complex in which hydrophobic extensions on the proteins serve to maintain them in their high molecular weight form, but are not needed for catalytic activity. The structural similarity of these enzymes to certain membrane-bound proteins, and its implications for synthetase localization and function in vivo, are discussed. PMID:6746733

  11. Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro (Tokyo Univ. (Japan). Faculty of Science)

    1984-03-01

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both (1-/sup 14/C)-acetate and (2/sup 14/C) malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases.

  12. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

    Directory of Open Access Journals (Sweden)

    Medina Milagros

    2008-09-01

    Full Text Available Abstract 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.

  13. Glutamine versus ammonia utilization in the NAD synthetase family.

    Directory of Open Access Journals (Sweden)

    Jessica De Ingeniis

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

  14. The Mitochondrial Aminoacyl tRNA Synthetases: Genes and Syndromes

    Directory of Open Access Journals (Sweden)

    Daria Diodato

    2014-01-01

    Full Text Available Mitochondrial respiratory chain (RC disorders are a group of genetically and clinically heterogeneous diseases. This is because protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis and maintenance of mitochondria, including mitochondrial DNA (mtDNA replication, transcription, and translation, require nuclear-encoded genes. In the past decade, a growing number of syndromes associated with dysfunction of mtDNA translation have been reported. This paper reviews the current knowledge of mutations affecting mitochondrial aminoacyl tRNAs synthetases and their role in the pathogenic mechanisms underlying the different clinical presentations.

  15. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...... groups of rats. These changes may partly explain the demonstrated training-induced increase in glucose tolerance. None of the findings could be ascribed to differences in foold intake or body weight....

  16. Primer Dependent and Independent Forms of Soluble Starch Synthetase from Developing Barley Endosperms

    DEFF Research Database (Denmark)

    Kreis, M.

    1980-01-01

    The activity of soluble starch synthetase (ADP-glucose: agr-1,4-glucan agr-4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purif...

  17. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...

  18. Uroporphyrinogen-I-synthetase activity in red blood cells of lead-exposed workers

    Energy Technology Data Exchange (ETDEWEB)

    El-Waseef, A.

    1982-01-01

    Lead-exposed (n . 26) and control (n . 12) subjects were investigated for their blood lead concentration erythrocyte 5-amino-laevulinic acid dehydratase (5-ALAD) and erythrocyte uroporphyrinogen-I-synthetase (URO-I-S) activity; 5-amino-laevulinic acid (5-ALA) and porphobilinogen (PBG) were used as substrates in the synthetase assay. In the lead workers erythrocyte 5-ALA dehydratase was grossly inhibited but with PBG as substrate the synthetase activity was not significantly different from the control group. With 5-ALA as substrate the synthetase assay showed marked inhibition. Addition of zinc (0.1 mmol/l) and dithiotheritol (0.5 mmol/l) brought the activities of both the dehydratase and synthetase (using 5-ALA as substrate) back into the ranges seen in the control group. With porphobilinogen as substrate higher concentrations of zinc caused inhibition of the synthetase, whilst reduction of added zinc to 0.01 mmol/l resulted in stimulation of the synthetase. A good correlation (r . 0.87) was obtained in synthetase assay when PBG and 5-aminolaevulinate (with added zinc and dithiothreitol) were used as substrates. With these additions 5-ALA may be used as a substrate in the URO-I-S assay in the investigation of latent cases of acute intermittent porphyria.

  19. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  20. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    Energy Technology Data Exchange (ETDEWEB)

    Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

    2016-03-15

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  1. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  2. Knockdown of asparagine synthetase (ASNS) suppresses cell proliferation and inhibits tumor growth in gastric cancer cells.

    Science.gov (United States)

    Yu, Qingxiang; Wang, Xiaoyu; Wang, Li; Zheng, Jia; Wang, Jiang; Wang, Bangmao

    2016-10-01

    Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine. ASNS is deemed as a promising therapeutic target and its expression is associated with the chemotherapy resistance in several human cancers. However, its role in gastric cancer tumorigenesis has not been investigated. In this study, we employed small interfering RNA (siRNA) to transiently knockdown ASNS in two gastric cancer cell lines, AGS and MKN-45, followed by growth rate assay and colony formation assay. Dose response curve analysis was performed in AGS and MKN-45 cells with stable ASNS knockdown to assess sensitivity to cisplatin. Xenograft experiment was performed to examine in vivo synergistic effects of ASNS depletion and cisplatin on tumor growth. Expression level of ASNS was evaluated in human patient samples using quantitative PCR. Kaplan-Meier curve analysis was performed to evaluate association between ASNS expression and patient survival. Transient knockdown of ASNS inhibited cell proliferation and colony formation in AGS and MKN-45 cells. Stable knockdown of ASNS conferred sensitivity to cisplatin in these cells. Depletion of ASNS and cisplatin treatment exerted synergistic effects on tumor growth in AGS xenografts. Moreover, ASNS was found to be up-regulated in human gastric cancer tissues compared with matched normal colon tissues. Low expression of ASNS was significantly associated with better survival in gastric cancer patients. ASNS may contribute to gastric cancer tumorigenesis and may represent a novel therapeutic target for prevention or intervention of gastric cancer.

  3. The Sites of Transcription and Translation for Euglena Chloroplastic Aminoacyl-tRNA Synthetases

    Science.gov (United States)

    Hecker, L. I.; Egan, J.; Reynolds, R. J.; Nix, C. E.; Schiff, J. A.; Barnett, W. Edgar

    1974-01-01

    We find that cycloheximide completely blocks the light-induced apearance of Euglena chloroplastic aminoacyl-tRNA synthetases in dark-grown cells of Euglena gracilis var. bacillaris. Streptomycin, on the other hand, has no effect on the light-induction of these organellar enzymes. These observations, together with the finding that an aplastidic mutant (strain W3BUL, which has neither significant plastid structure nor detectable chloroplast DNA) contains low levels of the chloroplastic synthetases, indicate that the chloroplastic synthetases are transcriptional products of nuclear genes and are translated on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts. PMID:4525469

  4. In Silico Discovery of Aminoacyl-tRNA Synthetase Inhibitors

    Directory of Open Access Journals (Sweden)

    Yaxue Zhao

    2014-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNA. They play a pivotal role in protein synthesis and are essential for cell growth and survival. The aaRSs are one of the leading targets for development of antibiotic agents. In this review, we mainly focused on aaRS inhibitor discovery and development using in silico methods including virtual screening and structure-based drug design. These computational methods are relatively fast and cheap, and are proving to be of great benefit for the rational development of more potent aaRS inhibitors and other pharmaceutical agents that may usher in a much needed generation of new antibiotics.

  5. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

    Directory of Open Access Journals (Sweden)

    Adam C. Mirando

    2014-12-01

    Full Text Available 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.

  6. Common peptides study of aminoacyl-tRNA synthetases.

    Directory of Open Access Journals (Sweden)

    Assaf Gottlieb

    Full Text Available BACKGROUND: Aminoacyl tRNA synthetases (aaRSs constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains. RESULTS: We utilized the Common Peptides (CPs framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS-class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA enzyme overlapping binding sites in both families. CONCLUSIONS: The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.

  7. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward M. [Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics

    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+ 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+ 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+ 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)

  8. Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase

    Directory of Open Access Journals (Sweden)

    Liron Klipcan

    2010-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS—bacterial (αβ2, eukaryotic/archaeal cytosolic (αβ2, and mitochondrial α—is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNAPhe anticodon recognition.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency : Implications for treatment?

    NARCIS (Netherlands)

    van Spronsen, F. J.; Reijngoud, D. J.; Verhoeven, N. M.; Soorani-Lunsing, R. J.; Jakobs, C.; Sijens, P. E.

    2006-01-01

    Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was

  11. Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

    CSIR Research Space (South Africa)

    Salisu, S

    2011-05-01

    Full Text Available In research directed at the development of adenine triphosphate (ATP) analogs as potential glutamine synthetase (GS) inhibitors, adenine and allopurinol derivatives have been synthesized either as novel ATP analogs or as scaffolds...

  12. 3-substituted anilines as scaffolds for the construction of glutamine synthetase and DXP-reductoisomerase inhibitors

    CSIR Research Space (South Africa)

    Mutorwa, M

    2009-01-01

    Full Text Available -1 Synthetic Communications Volume 39, Issue 15, 2009 3-Substituted Anilines as Scaffolds for the Construction of Glutamine Synthetase and DXP-Reductoisomerase Inhibitors Marius Mutorwaa, Sheriff Salisua, Gregory L. Blatchbc, Colin Kenyond & Perry T...

  13. Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

    NARCIS (Netherlands)

    Lamers, W. H.; van Roon, M.; Mooren, P. G.; de Graaf, A.; Charles, R.

    1985-01-01

    A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic

  14. Holocarboxylase Synthetase 1 Physically Interacts with Histone H3 in Arabidopsis

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    Xi Chen

    2013-01-01

    Full Text Available Biotin is a water-soluble vitamin required by all organisms, but only synthesized by plants and some bacterial and fungal species. As a cofactor, biotin is responsible for carbon dioxide transfer in all biotin-dependent carboxylases, including acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, and pyruvate carboxylase. Adding biotin to carboxylases is catalyzed by the enzyme holocarboxylase synthetase (HCS. Biotin is also involved in gene regulation, and there is some indication that histones can be biotinylated in humans. Histone proteins and most histone modifications are highly conserved among eukaryotes. HCS1 is the only functional biotin ligase in Arabidopsis and has a high homology with human HCS. Therefore, we hypothesized that HCS1 also biotinylates histone proteins in Arabidopsis. A comparison of the catalytic domain of HCS proteins was performed among eukaryotes, prokaryotes, and archaea, and this domain is highly conserved across the selected organisms. Biotinylated histones could not be identified in vivo by using avidin precipitation or two-dimensional gel analysis. However, HCS1 physically interacts with Arabidopsis histone H3 in vitro, indicating the possibility of the role of this enzyme in the regulation of gene expression.

  15. Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

    Science.gov (United States)

    León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

    2017-08-21

    The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-11

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

  17. The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

    Directory of Open Access Journals (Sweden)

    Ariel Alperstein

    Full Text Available The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

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

    Science.gov (United States)

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

    2017-08-15

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

  19. Epilepsy in glioblastoma multiforme: correlation with glutamine synthetase levels.

    Science.gov (United States)

    Rosati, Anna; Marconi, Silvia; Pollo, Bianca; Tomassini, Alessia; Lovato, Laura; Maderna, Emanuela; Maier, Klaus; Schwartz, Andreas; Rizzuto, Nicolò; Padovani, Alessandro; Bonetti, Bruno

    2009-07-01

    The hypothesis addressed by this study is that a glutamine synthetase (GS) deficiency in neoplastic astrocytes is a possible molecular basis associated with seizure generation in glioblastoma multiforme (GBM). Quantitative Western blot analysis of GS was performed in 20 individuals operated for malignant glioma. The levels of GS in patients with GBM and epilepsy were significantly lower (range 0.04-1.15; mean 0.35 +/- 0.36; median 0.25) than in non-epileptic GBM individuals (range 0.78-3.97; mean 1.64 +/- 0.99; median 1.25; P = 0.002). No relationship has been found between histological features (i.e. necrosis, gliosis, stroma, inflammatory cells, giant cells, and haemosiderine) and GS expression or epilepsy. Even though the epileptogenesis in glioma is multifactorial, it is conceivable that a down-regulation of GS may have an important pro-epileptogenic role in GBM, through the slowing of glutamate-glutamine cycle. This study suggests that seizures in GBM are coupled with a highly localized enzyme deficiency. The manipulation of GS activity might constitute a novel principle for inhibiting seizures in patients with glioma epilepsy.

  20. Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

    LENUS (Irish Health Repository)

    Kell, M R

    2012-02-03

    BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

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

    Science.gov (United States)

    Palmieri, Erika M; Menga, Alessio; Lebrun, Aurore; Hooper, Douglas C; Butterfield, D Allan; Mazzone, Massimiliano; Castegna, Alessandra

    2017-03-10

    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. 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. 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. 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.

  2. Induction of glutamine synthetase and transient co-expression with carbamoylphosphate synthetase in hepatocytes transplanted into fat pads of syngeneic hosts

    NARCIS (Netherlands)

    Gebhardt, R.; Jirtle, R.; Moorman, A. F.; Lamers, W. H.; Michalopoulos, G.

    1989-01-01

    Isolated rat hepatocytes were transplanted into the interscapular and both anterior lateral fat pads of hepatectomized syngeneic rats. At various time points following transplantation, the fat pads were removed, fixed and embedded in paraffin. Serial sections were stained for glutamine synthetase

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

    Science.gov (United States)

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

    2003-01-01

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

  4. Substrate activation and conformational dynamics of guanosine 5'-monophosphate synthetase.

    Science.gov (United States)

    Oliver, Justin C; Linger, Rebecca S; Chittur, Sridar V; Davisson, V Jo

    2013-08-06

    Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.

  5. [Methionine sulfoximine and phosphinothricin--glutamine synthetase inhibitors and activators and their herbicidal activity (A review)].

    Science.gov (United States)

    Evstigneeva, Z G; Solov'eva, N A; Sidel'nikova, L I

    2003-01-01

    Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairments of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in a concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.

  6. Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-β-lactam.

    Science.gov (United States)

    Patrick, Garrett J; Fang, Luting; Schaefer, Jacob; Singh, Sukrit; Bowman, Gregory R; Wencewicz, Timothy A

    2018-01-09

    Tabtoxinine-β-lactam (TβL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TβL 3-(S)-hydroxy-β-lactam (3-HβL) warhead. Phosphorylation of TβL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TβL β-lactam ring remains intact during enzyme inhibition, making TβL mechanistically distinct from traditional β-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HβL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

    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

    2010-10-01

    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-x₂₂-C-x₆-C-x₂-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.

  9. A bacterial ortholog of class II lysyl-tRNA synthetase activates lysine.

    Science.gov (United States)

    Ambrogelly, Alexandre; O'Donoghue, Patrick; Söll, Dieter; Moses, Sarath

    2010-07-16

    Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs, essential substrates for accurate protein synthesis. Beyond their central role in translation some of these enzymes or their orthologs are recruited for alternative functions, not always related to their primary cellular role. We investigate here the enzymatic properties of GenX (also called PoxA and YjeA), an ortholog of bacterial class II lysyl-tRNA synthetase. GenX is present in most Gram-negative bacteria and is homologous to the catalytic core of lysyl-tRNA synthetase, but it lacks the amino terminal anticodon binding domain of the latter enzyme. We show that, in agreement with its well-conserved lysine binding site, GenX can activate in vitro l-lysine and lysine analogs, but does not acylate tRNA(Lys) or other cellular RNAs. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

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

    Science.gov (United States)

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

    1991-01-01

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

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

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

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

    Science.gov (United States)

    1983-07-01

    ammonia into urea in marine Chondrichthyes liver 7 Table 1--Liver and brain glutamine synthetase of urea-retaining and non-urea-retaining amphibians 8... Osteichthyes , is a marine fish that also retains urea as an osmolyte (3,12). It too has a relatively high level of glu- tamine synthetase in its liver (16...for assimilation of ammonia into urea in marine Chondrichthyes liver (from Webb (15)) C1 klA IWE LO, AT? VH3 tLt TKATICAkMUIYL PUOSPULArL I CflALLLINE

  14. A Bacterial Ortholog of Class II Lysyl-tRNA Synthetase Activates Lysine

    OpenAIRE

    Ambrogelly, Alexandre; O’Donoghue, Patrick; Söll, Dieter; Moses, Sharath

    2010-01-01

    Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs, essential substrates for accurate protein synthesis. Beyond their central role in translation some of these enzymes or their orthologs are recruited for alternative functions, not always related to their primary cellular role. We investigate here the enzymatic properties of GenX (also called PoxA and YjeA), an ortholog of bacterial class II lysyl-tRNA synthetase. GenX is present in most Gram-negative bacteria and is homologous to the catalyt...

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

    Science.gov (United States)

    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

  16. Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium Aphanizomenon flos-aquae.

    Science.gov (United States)

    Saker, M L; Jungblut, A-D; Neilan, B A; Rawn, D F K; Vasconcelos, V M

    2005-10-01

    In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1--4.72 microgg(-1) (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.

  17. The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

    Science.gov (United States)

    Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

    2017-07-18

    Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNAHis binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

  18. Genetic association of long-chain acyl-CoA synthetase 1 variants with fasting glucose, diabetes, and subclinical atherosclerosis.

    Science.gov (United States)

    Manichaikul, Ani; Wang, Xin-Qun; Zhao, Wei; Wojczynski, Mary K; Siebenthall, Kyle; Stamatoyannopoulos, John A; Saleheen, Danish; Borecki, Ingrid B; Reilly, Muredach P; Rich, Stephen S; Bornfeldt, Karin E

    2016-03-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) converts free fatty acids into acyl-CoAs. Mouse studies have revealed that ACSL1 channels acyl-CoAs to β-oxidation, thereby reducing glucose utilization, and is required for diabetes-accelerated atherosclerosis. The role of ACSL1 in humans is unknown. We therefore examined common variants in the human ACSL1 locus by genetic association studies for fasting glucose, diabetes status, and preclinical atherosclerosis by using the MAGIC and DIAGRAM consortia; followed by analyses in participants from the Multi-Ethnic Study of Atherosclerosis, the Penn-T2D consortium, and a meta-analysis of subclinical atherosclerosis in African Americans; and finally, expression quantitative trait locus analysis and identification of DNase I hypersensitive sites (DHS). The results show that three SNPs in ACSL1 (rs7681334, rs735949, and rs4862423) are associated with fasting glucose or diabetes status in these large (>200,000 subjects) data sets. Furthermore, rs4862423 is associated with subclinical atherosclerosis and coincides with a DHS highly accessible in human heart. SNP rs735949 is in strong linkage disequilibrium with rs745805, significantly associated with ACSL1 levels in skin, suggesting tissue-specific regulatory mechanisms. This study provides evidence in humans of ACSL1 SNPs associated with fasting glucose, diabetes, and subclinical atherosclerosis and suggests links among these traits and acyl-CoA synthesis. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  19. ISOLEUCYL-tRNA-SYNTHETASE A FLUORESCENCE STUDY OP THE BINDINGPROPERTIES OF THE SYNTHETASE FROM ESCHERICHIA COLI

    Energy Technology Data Exchange (ETDEWEB)

    Penzer, Geoffrey R.; Bennett, Edward L.; Calvin, Melvin.

    1970-11-01

    Fluorescence properties of purified isoleucyl-tRNA-synthetase isolated from E. coli B have been studied. No changes in the quantum yield, energy or polarization of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L-isoleucine. In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme-ligand complexes. Values have been obtained for the complexes between enzyme and L-isoleucine (K{sub diss} = 2.5 x 10{sup -5} M), L-valine (K{sub diss} = 3.0 x 10{sup -4} M), ATP (K{sub diss} = 1.5 x 10{sup -4} M) and PP{sub i} (K{sub diss} = 2.0 x 10{sup -4} M) at 25{sup o}. The effects of ionic strength, and the temperature dependence and urea concentration dependence of L-isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and L-isoleucine together. The magnesium ion concentration dependence of this effect (half-point about 2 x 10{sup -4} M) and the equilibrium constant for L-isoleucine activation (2 x 10{sup -6} M) have both been measured. The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound L-isoleucylarenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.

  20. Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

    1989-01-01

    Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine repre...

  1. The parallel and convergent universes of polyketide synthases and nonribosomal peptide synthetases.

    Science.gov (United States)

    Cane, D E; Walsh, C T

    1999-12-01

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) catalyze chain elongation from simple building blocks to create a diverse array of natural products. PKS and NRPS proteins share striking architectural and organizational similarities that can be exploited to generate entirely new natural products.

  2. Isolation and characterization of the rat glutamine synthetase-encoding gene

    NARCIS (Netherlands)

    van de Zande, L.; Labruyère, W. T.; Arnberg, A. C.; Wilson, R. H.; van den Bogaert, A. J.; Das, A. T.; van Oorschot, D. A.; Frijters, C.; Charles, R.; Moorman, A. F.

    1990-01-01

    From a rat genomic library in phage lambda Charon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5-10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-length cDNAs containing a

  3. Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes

    NARCIS (Netherlands)

    van Roon, M. A.; Charles, R.; Lamers, W. H.

    1987-01-01

    Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such

  4. Functions of Glutamine Synthetase Isoforms in the Nitrogen Metabolism of Plants

    DEFF Research Database (Denmark)

    Guan, Miao

    ;2 which encode different isoforms of the key N-assimilatory enzyme cytosolic glutamine synthetase (GS1). In the single knockout mutant gln1;2 and in the double knockout mutant gln1;1:gln1;2, seed germination and seedling establishment were distinctly impaired. The negative effect of Gln1;2 deficiency...

  5. Regulation of the spatiotemporal pattern of expression of the glutamine synthetase gene

    NARCIS (Netherlands)

    Lie-Venema, H.; Hakvoort, T. B.; van Hemert, F. J.; Moorman, A. F.; Lamers, W. H.

    1998-01-01

    Glutamine synthetase, the enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, is expressed in a tissue-specific and developmentally controlled manner. The first part of this review focuses on its spatiotemporal pattern of expression, the factors that regulate

  6. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  7. Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

    NARCIS (Netherlands)

    Wagemaker, M.J.M.; Eastwood, D.C.; Drift, van der C.; Jetten, M.S.M.; Burton, K.; Griensven, van L.J.L.D.; Camp, op den H.J.M.

    2007-01-01

    Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main

  8. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid...

  9. ISOLATION AND CHARACTERIZATION OF THE RAT GENE FOR CARBAMOYLPHOSPHATE SYNTHETASE-I

    NARCIS (Netherlands)

    VANDENHOFF, MJB; VANDEZANDE, LPWGM; DINGEMANSE, MA; DAS, AT; LABRUYERE, W; MOORMAN, AFM; CHARLES, R; LAMERS, WH; Jacobus Mgn Van De Zande, Louis

    1995-01-01

    Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene

  10. Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli.

    Science.gov (United States)

    Snustad, D P; Hunsperger, J P; Chereskin, B M; Messing, J

    1988-12-01

    Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.

  11. Inhibition of glutathione synthesis in the liver leads to S-adenosyl-L-methionine synthetase reduction.

    Science.gov (United States)

    Corrales, F; Ochoa, P; Rivas, C; Martin-Lomas, M; Mato, J M; Pajares, M A

    1991-09-01

    The hepatic levels of glutathione in rats treated with buthionine sulfoximine (4 mmol/kg), an inhibitor of glutathione synthesis, were 72.5% +/- 4.9% of those determined in control animals. This decrease in glutathione concentration was prevented by the administration of glutathione monoethyl ester (7.5 mmol/kg). S-Adenosyl-L-methionine-synthetase activity in the liver of rats treated with buthionine sulfoximine was 39.4% +/- 6.5% of that determined in control animals. Again, glutathione monoethyl ester prevented the effect of buthionine sulfoximine on S-adenosyl-L-methionine-synthetase activity. There was a close correlation (r = 0.936) between the hepatic levels of glutathione and S-adenosyl-L-methionine-synthetase activity. The hepatic concentration of S-adenosyl-L-methionine in buthionine sulfoximine-treated animals was 59.7% +/- 3.7% of that measured in control rats. Contrasting with the protective effects mentioned above, glutathione monoester had no preventive action on buthionine sulfoximine-induced S-adenosyl-L-methionine depletion. Electron microscopic examination of liver samples of rats after buthionine sulfoximine administration showed evidence of liver degeneration, which was attenuated by glutathione monoethyl ester treatment. Glutathione (7.5 mmol/kg) treatment was less effective than glutathione monoethyl ester in attenuating buthionine sulfoximine effects on hepatic S-adenosyl-L-methionine metabolism and morphology. The reduction of S-adenosyl-L-methionine-synthetase activity observed after treatment with buthionine sulfoximine and its prevention by glutathione monoethyl ester, as well as the correlation between the activity of this enzyme and glutathione levels, indicate that glutathione plays an important role in maintaining S-adenosyl-L-methionine-synthetase activity in the liver.

  12. Molecular recognition of histidine tRNA by histidyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Nagatoyo, Yukari; Iwaki, Jun; Suzuki, Satoko; Kuno, Atsushi; Hasegawa, Tsunemi

    2005-01-01

    To investigate the recognition sites of histidine tRNA for histidyl-tRNA synthetase from an extreme hyperthermophilic archaeon, Aeropyrum pernix K1, we examined histidylation activities by using overexpressed histidyl-tRNA synthetase and various histidine tRNA transcripts that were prepared by in vitro transcription system. Results indicated that anticodon was not recognized by the histidyl-tRNA synthetase similar to that of Escherichia coli histidine tRNA recognition system. Discriminator base C73 was weekly recognized and an additional G residue was specifically recognized by the enzyme.

  13. Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis.

    Directory of Open Access Journals (Sweden)

    Arvind Sharma

    2016-11-01

    Full Text Available Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms and platyhelminths (also called flatworms. These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs. These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs are vital enzymes that decode genetic information and enable protein translation. The specific inhibition of pathogen aaRSs bores well for development of next generation anti-parasitics. Here, we have identified and annotated aaRSs and accessory proteins from Loa loa (nematode and Schistosoma mansoni (flatworm to provide a glimpse of these protein translation enzymes within these parasites. Using purified parasitic lysyl-tRNA synthetases (KRSs, we developed series of assays that address KRS enzymatic activity, oligomeric states, crystal structure and inhibition profiles. We show that L. loa and S. mansoni KRSs are potently inhibited by the fungal metabolite cladosporin. Our co-crystal structure of Loa loa KRS-cladosporin complex reveals key interacting residues and provides a platform for structure-based drug development. This work hence provides a new direction for both novel target discovery and inhibitor development against eukaryotic pathogens that include L. loa and S. mansoni.

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

    Science.gov (United States)

    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.

  15. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

    Science.gov (United States)

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

  16. Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: II. Arginosuccinate Synthetase and Lyase.

    Science.gov (United States)

    Roubelakis, K A; Kliewer, W M

    1978-09-01

    Arginosuccinate (ASA) synthetase and lyase activities were detected in extracts from Vitis vinifera L. cv. Chenin blanc mature leaves and seedlings. Optimum reaction conditions for ASA synthetase were 10 millimolar l-citrulline, 7.5 millimolar l-aspartate, 3 to 4 millimolar ATP, 12 millimolar Mg(2+) (pH 7.5 to 8.0), enzyme extract up to equivalent of about 200 milligrams of fresh tissue, and incubation temperature of 38 to 40 C. Optimum reaction conditions for ASA lyase were 4 millimolar ASA-K salt (pH 7.3 to 7.8), amount of extract up to equivalent of about 180 milligrams of fresh tissue, and incubation temperature of 38 to 40 C.

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

    Science.gov (United States)

    Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

    1987-01-01

    The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

  18. Fatty acid synthetase from Brevibacterium ammoniagenes: formation of monounsaturated fatty acids by a multienzyme complex.

    Science.gov (United States)

    Kawaguchi, A; Okuda, S

    1977-01-01

    A multienzyme fatty acid synthetase complex isolated from Brevibacterium ammoniagenes has been purified to a specific activity of 1440 nmol of malonyl-CoA incorporated per min/mg. The enzyme is homogeneous, as judged by gel electrophoresis on agarose gels, and has a molecular weight of 1.2 X 10(6). Both NADPH and NADH are required for activity. In contrast to other fatty acid synthetase complexes, the enzyme catalyzes the synthesis of both long-chain saturated and monounsaturated fatty acids from malonyl-CoA and acetyl-CoA. The formation of unsaturated fatty acids is oxygen-independent and sharply reduced by 3-decynoyl-N-acetylcysteamine, a known inhibitor of Escherchia coli beta-hydroxydecanoyl thioester dehydrase (EC 4.2.1.60). PMID:20622

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

    Science.gov (United States)

    1980-09-01

    glutamine synthetase activity is defined as the production of one pmole of y-glutamyl hydroxamate per min at 25°C. Protein was determined by the biuret method ...content. P. is listed as at progein per g tissue ( biuret method ); nm ± standard deviation.’ Number of specimens examined is listed in parenthesis. 3 body...lamprey, coelacanth . AOSTRACT (Continue en revere., olde It necessary and Identify by block nuotbor) nzyme assays using the y-glutamyl transferase method

  20. Pasteurella multocida CMP-sialic acid synthetase and mutants of Neisseria meningitidis CMP-sialic acid synthetase with improved substrate promiscuity.

    Science.gov (United States)

    Li, Yanhong; Yu, Hai; Cao, Hongzhi; Muthana, Saddam; Chen, Xi

    2012-03-01

    Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

  1. Differential inactivation of alfalfa nodule glutamine synthetases by tabtoxinine-. beta. -lactam. [Pseudomonas syringae

    Energy Technology Data Exchange (ETDEWEB)

    Knight, T.J.; Unkefer, P.J.

    1987-04-01

    The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated alfalfa plants results in an increase in N/sub 2/-fixation potential and growth, but a 40-50% decrease in nodule glutamine synthetase (GS) activity, as compared to nodulated control plants. Tabtoxinine-..beta..-Lactam an exocellular toxin produced by Pseudomonas syringae pv tabaci irreversibly inhibits glutamine synthetase. Partial purification of nodule GS by DEAE-cellulose chromatography reveals two enzyme forms are present (GS/sub n1/ and GS/sub n2/). In vitro inactivation of the two glutamine synthetases associated with the nodule indicates a differential sensitivity to T-..beta..-L. The nodule specific GS/sub n1/ is much less sensitive to T-..beta..-L than the GS/sub n2/ enzyme, which was found to coelute with the root enzyme (GS/sub r/). However, both GS/sub n1/ and GS/sub n2/ are rapidly inactivated by methionine sulfoximine, another irreversible inhibitor of GS.

  2. Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy.

    Science.gov (United States)

    Haselhorst, Thomas; Münster-Kühnel, Anja K; Stolz, Anita; Oschlies, Melanie; Tiralongo, Joe; Kitajima, Ken; Gerardy-Schahn, Rita; von Itzstein, Mark

    2005-02-11

    CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.

  3. Structure of the terminal PCP domain of the non-ribosomal peptide synthetase in teicoplanin biosynthesis.

    Science.gov (United States)

    Haslinger, Kristina; Redfield, Christina; Cryle, Max J

    2015-04-01

    The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis. © 2015 Wiley Periodicals, Inc.

  4. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

    DEFF Research Database (Denmark)

    Elo, Jenni M; Yadavalli, Srujana S; Euro, Liliya

    2012-01-01

    Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochon......Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding...... the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial...... was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial...

  5. [High efficiency of L-glutamine production by coupling genetic engineered bacterial glutamine synthetase with yeast alcoholic fermentation system].

    Science.gov (United States)

    Chen, Qun-Ying; Chen, Guo-An; Xue, Bin; Zhang, Xian-Jiu; Yin, Zhi-Min

    2004-05-01

    Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved

  6. Regulation of 3-Deoxy-d-arabino-Heptulosonic 7-Phosphate Acid Synthetase Activity in Relation to the Synthesis of the Aromatic Vitamins in Escherichia coli K-12

    Science.gov (United States)

    Wallace, B. J.; Pittard, J.

    1969-01-01

    Both in vivo and in vitro experiments on wild-type Escherichia coli K-12 and mutant strains possessing only single 3-deoxy-d-arabino-heptulosonic 7-phosphate acid (DAHP) synthetase isoenzymes indicated that, under conditions when all three isoenzymes are fully repressed, sufficient chorismate is still formed for the synthesis of aromatic vitamins. Under repressed conditions both DAHP synthetase (phe) and (trp), but not DAHP synthetase (tyr), were shown to contribute to vitamin production. PMID:4905534

  7. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.

    2006-01-01

    Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]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 synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored...... after the addition of exogenous wild-type mouse or human alpha-synuclein, but not by A30P, E46K, and A53T forms of alpha-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools were markedly reduced...

  8. Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

    Directory of Open Access Journals (Sweden)

    Pengfei Fang

    2015-12-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

  9. The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

    Science.gov (United States)

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

    2017-05-18

    Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Antiviral Mechanisms of the 2'-5' Oligoadenylate Synthetases

    DEFF Research Database (Denmark)

    Kristiansen, Helle

    During the course of her PhD studies, Helle Kristiansen carried out research into proteins that play a role in the defence against viral infection in humans and animals. All cells contain a considerable number of proteins that can directly or directly attack a virus and inhibit or completely stop...

  11. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.

    Science.gov (United States)

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V; Carvalho, Helena G; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-04-01

    The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  12. Effect of Mini-Tyrosyl-tRNA Synthetase/Mini-Tryptophanyl-tRNA Synthetase on Angiogenesis in Rhesus Monkeys after Acute Myocardial Infarction.

    Science.gov (United States)

    Zeng, Rui; Wang, Mian; You, Gui-ying; Yue, Rong-zheng; Chen, Yu-cheng; Zeng, Zhi; Liu, Rui; Qiang, Ou; Zhang, Li

    2016-02-01

    The purpose of this study was to clarify the effect of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase (mini-TyrRS/mini-TrpRS) in ischemic angiogenesis in rhesus monkeys with acute myocardial infarction (AMI). A 27-gauge needle was incorporated percutaneously into the left ventricular myocardium of rhesus monkeys with AMI. All monkeys were randomized to receive adenoviral vector mini-TyrRS/mini-TrpRS, which was administered as five injections into the infarcted myocardium, or saline or ad-null (control groups). The injections were guided by EnSite NavX left ventricular electroanatomical mapping. Mini-TyrRS/mini-TrpRS proteins were detected by Western blot and immunoprecipitation analyses. Microvessel density (MVD) per section was measured using immunostaining with a CD34 monoclonal antibody. Proliferating cardiomyocytes were identified through histological and immunohistochemical analyses. Myocardial perfusion and cardiac function were estimated by G-SPECT. Infarction size was also measured. Western blot analyses showed that compared to the normal zone, the expression level of mini-TyrRS/mini-TrpRS was significantly different in the infarction zone. G-SPECT analysis indicated that the mini-TyrRS group had better cardiac function and myocardial perfusion after the injection of ad-mini-TyrRS than before, while mini-TrpRS injection had a totally opposite effect. After mini-TyrRS was administered, there was less of an infarction zone and more proliferating cardiomyocytes and capillaries in the mini-TyrRS group compared to both of the control groups, and the ad-mini-TrpRS group had a totally opposite effect. These results indicated that angiogenesis could be either stimulated by mini-TyrRS or inhibited by mini-TrpRS. © 2015 John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    Anna Sivachenko

    2016-04-01

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

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

    Science.gov (United States)

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

    2016-04-01

    Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum(bgm) and double bubble(dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivois causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6(encoding a very-long-chain ACS), a human homolog of bgm and dbb. © 2016. Published by The Company of Biologists Ltd.

  15. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    Science.gov (United States)

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds.

    Science.gov (United States)

    Gaufichon, Laure; Marmagne, Anne; Belcram, Katia; Yoneyama, Tadakatsu; Sakakibara, Yukiko; Hase, Toshiharu; Grandjean, Olivier; Clément, Gilles; Citerne, Sylvie; Boutet-Mercey, Stéphanie; Masclaux-Daubresse, Céline; Chardon, Fabien; Soulay, Fabienne; Xu, Xiaole; Trassaert, Marion; Shakiebaei, Maryam; Najihi, Amina; Suzuki, Akira

    2017-08-01

    Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoterCaMV35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoterNapin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  17. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

    Directory of Open Access Journals (Sweden)

    Caroline Hoff-Risseti

    Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

  18. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    Science.gov (United States)

    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

  19. Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase.

    Science.gov (United States)

    Dulic, Morana; Cvetesic, Nevena; Zivkovic, Igor; Palencia, Andrés; Cusack, Stephen; Bertosa, Branimir; Gruic-Sovulj, Ita

    2017-10-27

    The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3'-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A 1H STD NMR spectroscopic investigation of sialylnucleoside mimetics as probes of CMP-Kdn synthetase.

    Science.gov (United States)

    Haselhorst, Thomas; Oschlies, Melanie; Abu-Izneid, Tareq; Kiefel, Milton J; Tiralongo, Joe; Münster-Kühnel, Anja K; Gerardy-Schahn, Rita; von Itzstein, Mark

    2006-07-01

    CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and CTP to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP( i ). Saturation Transfer Difference (STD) NMR spectroscopy has been employed to investigate the sub-structural requirements of the enzyme's binding domain. Sialylnucleoside mimetics, where the sialic acid moiety has been replaced by a carboxyl group and a hydrophobic moiety, have been used in NMR experiments, to probe the tolerance of the CMP-Kdn synthetase to such replacements. From our data it would appear that unlike another sialylnucleotide-recognising protein, the CMP-Neu5Ac transport protein, either a phosphate group or other functional groups on the sialic acid framework may play important roles in recognition by the synthetase.

  1. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  2. S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.

    Science.gov (United States)

    Corrales, F; Giménez, A; Alvarez, L; Caballería, J; Pajares, M A; Andreu, H; Parés, A; Mato, J M; Rodés, J

    1992-10-01

    Administration of carbon tetrachloride to rats resulted in induction of hepatic fibrosis and a 60% reduction of hepatic S-adenosylmethionine synthetase activity without producing any significant modification of hepatic levels of S-adenosylmethionine synthetase messenger RNA. The reduction of S-adenosylmethionine synthetase activity was corrected by treatment with S-adenosylmethionine (3 mg/kg/day, intramuscularly). Administration of carbon tetrachloride also produced a 45% depletion of liver glutathione (reduced form) that was corrected by S-adenosylmethionine treatment. After the rats received carbon tetrachloride, a 2.3-fold increase in liver collagen was observed; prolyl hydroxylase activity was 2.5 times greater than that seen in controls. These increases were attenuated in animals treated with carbon tetrachloride and S-adenosylmethionine. The attenuation by S-adenosylmethionine treatment of the fibrogenic effect of carbon tetrachloride was associated with a decrease in the number of rats in which cirrhosis developed.

  3. Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

    Science.gov (United States)

    Bolivar, F; Galván, M; Martuscelli, J

    1976-05-01

    An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.

  4. Diversity of nonribosomal peptide synthetase genes in the microbial metagenomes of marine sponges.

    Science.gov (United States)

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

    2012-06-01

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

  5. PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carter, Andrew T.; Beiche, Flora; Hove-Jensen, Bjarne

    1997-01-01

    In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS......) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has...

  6. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J

    2012-01-01

    )-triphosphatase 205, thiamine monophosphate kinase 179, pyruvate formate lyase family activating protein 298, 3-hydroxy-3-methylglutaryl-CoA reductase (mevanolate), N(2), N(2)-dimethylguanosine tRNA methyltransferase 145, N2, N2-dimethylguanosine tRNA methyltransferase 170, putative 5-methylcytosine restriction......-oxoglutarate ferredoxin oxidoreductase subunit gamma 352, 2-oxoglutarate ferredoxin oxidoreductase subunit alpha 407, methylmalonyl-CoA mutase, N-terminus of large subunit 172, AP endonuclease (base excision repair pathway) 365, CTP synthetase 105, PBP family phospholipid-binding protein 272, lipoate...

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

    Science.gov (United States)

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

    2008-01-01

    We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount...

  8. Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis.

    Science.gov (United States)

    Gantt, J S; Arfin, S M

    1981-07-25

    The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.

  9. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  10. Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1988-01-01

    A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histi......-less strain and suggest that PRPP synthetase is dispensable for E. coli....

  11. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adeno......The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...... catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction....

  12. Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Theilgaard, Hanne Birgitte; Kristiansen, K.N.; Henriksen, Claus Maxel

    1997-01-01

    delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)(2)SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The mole......delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)(2)SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography...

  13. Neurospora crassa glutamine synthetase. Role of enzyme synthesis and degradation on the regulation of enzyme concentration during exponential growth.

    Science.gov (United States)

    Quinto, C; Mora, J; Palacios, R

    1977-12-10

    The specific activity of Neurospora crassa glutamine synthetase varies according to the nitrogen source in which the organism is grown. In a poor nitrogen source such as glutamate, the specific activity of the enzyme is higher than that found in good nitrogen sources such as ammonium or glutamine. These differences in specific enzyme activity correspond to differences in enzyme concentration. The relative rates of glutamine synthetase synthesis and degradation were measured in exponential cultures grown in different nitrogen sources. The differences in enzyme concentration are explained by differences in the relative rate of enzyme synthesis.

  14. Recognition sites of glycine tRNA for glycyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Okamoto, Koji; Kuno, Atsushi; Hasegawa, Tsunemi

    2005-01-01

    To elucidate the tRNA recognition sites of glycine tRNA from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, we examined glycylation activities using in vitro mutant glycine tRNA transcripts and recombinant A. pernix glycyl-tRNA synthetase. The recognition nucleotides were determined to be C35 and C36 of anticodon, C2-G71 and G3-C70 base-pairs of acceptor stem. However, discriminator base A73 was not recognized by glycyl-tRNA synthetase.

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

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

    2012-01-01

    of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased......The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product...

  16. A Major Determinant for Binding and Aminoacylation of tRNAAla in Cytoplasmic Alanyl-tRNA Synthetase Is Mutated in Dominant Axonal Charcot-Marie-Tooth Disease

    Science.gov (United States)

    Latour, Philippe; Thauvin-Robinet, Christel; Baudelet-Méry, Chantal; Soichot, Pierre; Cusin, Veronica; Faivre, Laurence; Locatelli, Marie-Claire; Mayençon, Martine; Sarcey, Annie; Broussolle, Emmanuel; Camu, William; David, Albert; Rousson, Robert

    2010-01-01

    Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at θ = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNAAla aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features. PMID:20045102

  17. A Drosophila model for mito-nuclear diseases generated by an incompatible interaction between tRNA and tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Marissa A. Holmbeck

    2015-08-01

    Full Text Available Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes, which produce the majority of cellular energy, requires the coordinated expression and translation of both mitochondrially and nuclear-encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations both in mitochondrial DNA (mtDNA- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, we characterized a dual genome epistasis in which a naturally occurring mutation in the Drosophila simulans simw501 mtDNA-encoded transfer RNA (tRNA for tyrosine (tRNATyr interacts with a mutation in the nuclear-encoded mitochondrially localized tyrosyl-tRNA synthetase from Drosophila melanogaster. Here, we show that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased oxidative phosphorylation (OXPHOS enzyme activity and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw501 mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction might be tissue specific. Because mutations in mitochondrial tRNATyr are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other, mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function.

  18. 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

    Science.gov (United States)

    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.

    2013-01-01

    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. PMID:26343849

  19. Genetic Diversity of Dihydropteroate synthetase Gene (dhps Of Plasmodium vivax in Hormozgan Province, Iran

    Directory of Open Access Journals (Sweden)

    Somayeh MAGHSOODLOORAD

    2016-03-01

    Full Text Available Background: The present study was formulated in order to determine pol­ymorphism of dihydropteroate synthetase gene (dhps of Plasmodium vivax (P. vivax in Hormozgan Province, southern Iran and mutations at codons 382, 383, 512, 553, and 585 associated with resistance of P. vivax to sulfadoxine.Method: One-hundred eighteen isolates of P. vivax were prepared within 2007-2008 to determine dihydrofolate reductase-thymidylate synthase (dhfr-ts gene. The isolates were determined in the study of genetic diversity of dihy­dropteroate synthetase gene (dhps of P. vivax. The study was performed via PCR test and nucleotide sequencing.Results: Of 118 blood samples infected by P. vivax, 46 and 72 samples be­longed to Minab and Jask, respectively. No mutation was detected at 5 target codons. However, among these 118 samples, three isolates (2.54% were found to have a mutation at the new codon 421.Conclusion: Since mutation was detected in dihydrofolate reductase (Pvdhfr gene in the same samples but no mutation was found at five main codons of Pvdhps gene, it can be concluded that P. vivax, considering their mutations in Pvdhfr, is still susceptible to sulfadoxine and therefore, to fansidar in Hor­mozgan Province, Southern Iran.

  20. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  1. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    Science.gov (United States)

    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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

    Science.gov (United States)

    Khan, Sameena; Garg, Ankur; Sharma, Arvind; Camacho, Noelia; Picchioni, Daria; Saint-Léger, Adélaïde; de Pouplana, Lluís Ribas; Yogavel, Manickam; Sharma, Amit

    2013-01-01

    Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations. PMID:23776638

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

    Directory of Open Access Journals (Sweden)

    Ana Rita Seabra

    2015-07-01

    Full Text Available Glutamine Synthetase (GS catalyses 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 glutamine synthetase 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.

  4. Regulation of Nodule Glutamine Synthetase by CO2 Levels in Bean (Phaseolus vulgaris L.) 1

    Science.gov (United States)

    Ortega, José-Luis; Sánchez, Federico; Soberón, Mario; Flores, Miguel Lara

    1992-01-01

    Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO2 and from day 8 to 17 in high (0.1%) CO2 in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO2 compared with plants grown in 0.02% CO2 and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO2, particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO2 did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the γ polypeptide was significantly reduced in response to high CO2, whereas the β polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper. ImagesFigure 4 PMID:16668681

  5. Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene.

    Science.gov (United States)

    Meussen, Bas J; Weusthuis, Ruud A; Sanders, Johan P M; Graaff, Leo H de

    2012-02-01

    Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.

  6. Compound heterozygous mutations in glycyl-tRNA synthetase (GARS cause mitochondrial respiratory chain dysfunction.

    Directory of Open Access Journals (Sweden)

    Michael Nafisinia

    Full Text Available Glycyl-tRNA synthetase (GARS; OMIM 600287 is one of thirty-seven tRNA-synthetase genes that catalyses the synthesis of glycyl-tRNA, which is required to insert glycine into proteins within the cytosol and mitochondria. To date, eighteen mutations in GARS have been reported in patients with autosomal-dominant Charcot-Marie-Tooth disease type 2D (CMT2D; OMIM 601472, and/or distal spinal muscular atrophy type V (dSMA-V; OMIM 600794. In this study, we report a patient with clinical and biochemical features suggestive of a mitochondrial respiratory chain (MRC disorder including mild left ventricular posterior wall hypertrophy, exercise intolerance, and lactic acidosis. Using whole exome sequencing we identified compound heterozygous novel variants, c.803C>T; p.(Thr268Ile and c.1234C>T; p.(Arg412Cys, in GARS in the proband. Spectrophotometric evaluation of the MRC complexes showed reduced activity of Complex I, III and IV in patient skeletal muscle and reduced Complex I and IV activity in the patient liver, with Complex IV being the most severely affected in both tissues. Immunoblot analysis of GARS protein and subunits of the MRC enzyme complexes in patient fibroblast extracts showed significant reduction in GARS protein levels and Complex IV. Together these studies provide evidence that the identified compound heterozygous GARS variants may be the cause of the mitochondrial dysfunction in our patient.

  7. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

    Science.gov (United States)

    Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  8. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  9. Organ-specific activity of the 5' regulatory region of the glutamine synthetase gene in developing mice

    NARCIS (Netherlands)

    Lie-Venema, H.; de Boer, P. A.; Moorman, A. F.; Lamers, W. H.

    1997-01-01

    Glutamine synthetase (GS) converts ammonia and glutamate into glutamine. We assessed the activity of the 5' regulatory region of the GS gene in developing transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of 3150 bp of the upstream sequence of the rat GS

  10. Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity inEscherichia coli.

    Science.gov (United States)

    Ye, Qing; Ji, Quan-Quan; Yan, Wei; Yang, Fang; Wang, En-Duo

    2017-06-23

    Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetylation of Lys. In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli ( Ec LeuRS and Ec ArgRS) were overexpressed and purified and found to be acetylated on Lys residues by MS. Gln scanning mutagenesis revealed that Lys 619 , Lys 624 , and Lys 809 in Ec LeuRS and Lys 126 and Lys 408 in Ec ArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated Lys at specific sites and investigated their catalytic activity. Acetylation of these Lys residues could affect their aminoacylation activity by influencing amino acid activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate nonenzymatically acetylates Ec LeuRS and Ec ArgRS and suggested that the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aminoacyl-tRNA synthetases and thus protein synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation

    DEFF Research Database (Denmark)

    Banerjee, Rajat; Reynolds, Noah M; Yadavalli, Srujana S

    2011-01-01

    that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as aminoacyl-tRNA synthetases (aaRSs), can also lead to disease. In some cases, mutations can be directly linked to losses in enzymatic activity; however, for many, their effect is unknown. To investigate how aa...

  12. Organization and expression of genes in the genomic region surrounding the glutamine synthetase gene Gln1 from Lotus japonicus

    DEFF Research Database (Denmark)

    Thykjaer, T; Danielsen, D; She, Q

    1997-01-01

    within the 23326-bp genomic region analysed. The LjGln1 gene encodes a cytosolic glutamine synthetase and the LjKrm (Kinesin repeat motif) gene encodes a polypeptide with similarity to a repeated motif present in the microtubule-associated kinesin light chain protein. Transcripts of the glutamine...

  13. Purification and Properties of a Prokaryote Type Glutamine Synthetase from the Bialaphos Producer Streptomyces hygroscopicus SF1293

    NARCIS (Netherlands)

    Kumada, Yoichi; Takano, Eriko; Nagaoka, Kozo

    1990-01-01

    A prokaryote type glutamine synthetase (GS) was purified from a bialaphos (BA)-producing organism, Streptomyces hygroscopicus SF1293 (SF1293). The GS (GS I) consisted of a 55,000 dalton subunit, and its N-terminal amino acid sequence was similar to that of S. coelicolor GS. GS I was highly sensitive

  14. Novel expression pattern of cytosolic gln synthetase in nitrogen-fixing root nodules of the actinorhizal host, Datisca glomerata

    NARCIS (Netherlands)

    Berry, A.M.; Murphy, T.M.; Okubara, P.A.; Jacobsen, K.R.; Swensen, S.M.; Pawlowski, K.

    2004-01-01

    Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform,

  15. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown to b...

  16. Modulation of 2{prime}-5{prime} oligoadenylate synthetase by environmental stress in the marine sponge Geodia cydonium

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G. [Abteilung Angewandte Molekularbiologie, Mainz (Germany). Inst. fuer Physiologische Chemie; Kuusksalu, A.; Kelve, M. [Inst. of Chemical Physics and Biophysics, Tallinn (Estonia)

    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.

  17. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2. A w...... of long-chain fatty acids was synthesised from carboxylated acetyl-CoA than from added malonyl-CoA. - 5. It is suggested that acetyl-CoA carboxylase may carboxylate acetate bound to fatty acid synthetase.......- 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2....... A wide range (C4:0–C18:0) of fatty acids was synthesised and the proportions were modified by substrate concentrations in the same manner as for purified rabbit mammary gland fatty acid synthetase. - 3. The relative amount of radioactivity incorporated from added acetyl-CoA and malonyl-CoA depended...

  18. Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.

    2013-07-01

    Full Text Available In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA rather than usual 4 or 5 nucleotides (type 1 tRNA. In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong

  19. Role of aminoacyl-tRNA synthetases in infectious diseases and targets for therapeutic development.

    Science.gov (United States)

    Dewan, Varun; Reader, John; Forsyth, Karin-Musier

    2014-01-01

    Aminoacyl-tRNA synthetases (AARSs) play a pivotal role in protein synthesis and cell viability. These 22 "housekeeping" enzymes (1 for each standard amino acid plus pyrrolysine and o-phosphoserine) are specifically involved in recognizing and aminoacylating their cognate tRNAs in the cellular pool with the correct amino acid prior to delivery of the charged tRNA to the protein synthesis machinery. Besides serving this canonical function, higher eukaryotic AARSs, some of which are organized in the cytoplasm as a multisynthetase complex of nine enzymes plus additional cellular factors, have also been implicated in a variety of non-canonical roles. AARSs are involved in the regulation of transcription, translation, and various signaling pathways, thereby ensuring cell survival. Based in part on their versatility, AARSs have been recruited by viruses to perform essential functions. For example, host synthetases are packaged into some retroviruses and are required for their replication. Other viruses mimic tRNA-like structures in their genomes, and these motifs are aminoacylated by the host synthetase as part of the viral replication cycle. More recently, it has been shown that certain large DNA viruses infecting animals and other diverse unicellular eukaryotes encode tRNAs, AARSs, and additional components of the protein-synthesis machinery. This chapter will review our current understanding of the role of host AARSs and tRNA-like structures in viruses and discuss their potential as anti-viral drug targets. The identification and development of compounds that target bacterial AARSs, thereby serving as novel antibiotics, will also be discussed. Particular attention will be given to recent work on a number of tRNA-dependent AARS inhibitors and to advances in a new class of natural "pro-drug" antibiotics called Trojan Horse inhibitors. Finally, we will explore how bacteria that naturally produce AARS-targeting antibiotics must protect themselves against cell suicide using

  20. Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia

    Science.gov (United States)

    Furuyama, Kazumichi; Sassa, Shigeru

    2000-01-01

    The first and the rate-limiting enzyme of heme biosynthesis is δ-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the β subunit of human ATP-specific succinyl CoA synthetase (SCS-βA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-βA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-βA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-βA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria. PMID:10727444

  1. Determination of tryptophan tRNA recognition sites for tryptophanyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Tsuchiya, Wataru; Umehara, Takuya; Kuno, Atsushi; Hasegawa, Tsunemi

    2004-01-01

    To investigate the recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase from extreme hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, tryptophanylation activities were examined by using mutant tryptophan tRNA transcripts prepared by in vitro transcription system. Their transcripts were aminoacylated with tryptophan by overexpressed A. pernix tryptophanyl-tRNA synthetase. The results indicated that anticodon nucleotides C34, C35 and A36, discriminator base A73, G1-C72 and G2-C71 base pairs of acceptor stem were base-specifically recognized by A. pernix tryptophanyl-tRNA synthetase.

  2. Determination of phenylalanine tRNA recognition sites by phenylalanyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Tsuchiya, Wataru; Kimura, Manami; Hasegawa, Tsunemi

    2007-01-01

    Phenylalanine tRNA identity has been determined in the bacteria and the eukaryote system, but remains unknown for the archaea system. To investigate the molecular recognition mechanism of phenylalanine tRNA by phenylalanyl-tRNA synthetase from hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, various mutant transcripts of phenylalanine tRNA prepared by an in vitro transcription system were examined by overexpressed A. pernix phenylalanyl tRNA synthetase. The results indicated that anticodon nucleotides G34, A35 and A36, discriminator base A73 and G20 in the variable pocket were base-specifically recognized by A. pernix phenylalanyl-tRNA synthetase.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Marco Betti

    2012-06-01

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

  5. CMP-Sialic Acid Synthetase: The Point of Constriction in the Sialylation Pathway.

    Science.gov (United States)

    Sellmeier, Melanie; Weinhold, Birgit; Münster-Kühnel, Anja

    2015-01-01

    Sialoglycoconjugates form the outermost layer of animal cells and play a crucial role in cellular communication processes. An essential step in the biosynthesis of sialylated glycoconjugates is the activation of sialic acid to the monophosphate diester CMP-sialic acid. Only the activated sugar is transported into the Golgi apparatus and serves as a substrate for the linkage-specific sialyltransferases. Interference with sugar activation abolishes sialylation and is embryonic lethal in mammals. In this chapter we focus on the enzyme catalyzing the activation of sialic acid, the CMP-sialic acid synthetase (CMAS), and compare the enzymatic properties of CMASs isolated from different species. Information concerning the reaction mechanism and active site architecture is included. Moreover, the unusual nuclear localization of vertebrate CMASs as well as the biotechnological application of bacterial CMAS enzymes is addressed.

  6. Role of asparagine and asparagine synthetase genes in sunflower (Helianthus annuus) germination and natural senescence.

    Science.gov (United States)

    Herrera-Rodríguez, María Begoña; Maldonado, José María; Pérez-Vicente, Rafael

    2006-10-01

    Sunflower (Helianthus annuus) contains three active asparagine synthetase (EC 6.3.5.4, AS) genes: HAS1, HAS1.1 and HAS2. Asparagine content and AS gene expression were determined during germination and leaf and cotyledon natural senescence to assess the role of asparagine as well as the extent of participation of each AS gene in different nitrogen mobilizing processes. Asparagine accumulated in the dry seed and was the predominant amide throughout germination. During cotyledon senescence, the asparagine level was slightly higher than that of glutamine. The opposite was true for leaf senescence. According to transcript accumulation data, most of the asparagine newly synthesized for germination and cotyledon expansion was due to HAS2 activity, with little contribution of the other AS genes. However, all three genes work together to synthesize asparagine for leaf senescence. The absence of significant AS gene expression in cotyledon senescence differentiates leaf and cotyledon senescence, and suggests a cotyledon-specific regulation.

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

    Directory of Open Access Journals (Sweden)

    Ute Hentschel

    2012-05-01

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

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

    DEFF Research Database (Denmark)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne

    2014-01-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains...... of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine......, and aspartate and incorporation of (15)NH4(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS): discovery and perspectives.

    Science.gov (United States)

    Tahlan, Kapil; Moore, Marcus A; Jensen, Susan E

    2017-05-01

    The δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.

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

    Science.gov (United States)

    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. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  12. Purification, characterization, and expression of multiple glutamine synthetases from Prevotella ruminicola 23.

    Science.gov (United States)

    Kim, Jong Nam; Cann, Isaac K O; Mackie, Roderick I

    2012-01-01

    The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa. Optimal conditions for γ-glutamyl transferase activity were found to be 35°C at pH 5.6 with 0.25 mM Mn(2+) ions (GSI) or 37°C at pH 6.0 (GSIII-1 and GSIII-2) with 0.50 to 1.00 mM Mn(2+) ions. GSIII biosynthetic activity was found to be optimal at 50 to 60°C and pH 6.8 to 7.0 with 10 mM Mn(2+) ions, while GSI displayed no GS biosynthetic activity. Kinetic analysis revealed K(m) values for glutamate and ammonium as well as for hydrolysis of ATP to be 8.58, 0.48, and 1.91 mM, respectively, for GSIII-1 and 1.72, 0.43, and 2.65 mM, respectively, for GSIII-2. A quantitative reverse transcriptase PCR assay (qRT-PCR) revealed GSIII-2 to be significantly induced by high concentrations of ammonia, and this corresponded with increases in measured GS activity. Collectively, these results show that both GSIII enzymes in P. ruminicola 23 are functional and indicate that GSIII-2, flanked by GOGAT (gltB and gltD genes), plays an important role in the acquisition and metabolism of ammonia, particularly under nonlimiting ammonia growth conditions.

  13. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.

    2011-12-01

    Full Text Available Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa. Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates

  14. Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Wen; Jia, Haiyan; Zhang, Longmei; Wang, Haiyan; Tang, Hui; Zhang, Liping

    2017-08-01

    In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg2+/ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

  15. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...

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

    Science.gov (United States)

    Andrews, J; Keegstra, K

    1983-07-01

    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.

  17. Effect of anoxia and Polyscias filicifolia Bailey biomass tincture on the activity of tRNA and aminoacyl-tRNA synthetases in isolated pig heart.

    Science.gov (United States)

    Kasauskas, Artūras; Rodovicius, Hiliaras; Viezeliene, Dale; Lazauskas, Robertas

    2009-01-01

    The aim of this study was to investigate effect of anoxia and Polyscias filicifolia Bailey biomass tincture on the activities of different tRNA and aminoacyl-tRNA synthetases in isolated pig heart. The isolated pig heart was perfused according to the modified method of Langendorf, using an artificial blood circulation apparatus. Anoxia 20 min in duration was performed by perfusion of isolated heart with Krebs-Henseleit bicarbonate buffer saturated with gas mixture (95% N(2) and 5% CO(2)). Control heart was perfused with the same buffer saturated with gas mixture (95% O(2) and 5% CO(2)). Effect of Polyscias filicifolia Bailey biomass tincture was evaluated by perfusion of isolated heart with a buffer containing tincture. Total tRNA and aminoacyl-tRNA synthetases were isolated from pig heart. Activities of tRNA and aminoacyl-tRNA synthetases were measured by the aminoacylation reaction using C(14)-amino acids. Anoxia 20 min in duration has caused a decrease in the acceptor activity of tRNA and increase in the activities of aminacyl-tRNA synthetases. Polyscias filicifolia Bailey tincture did not affect the acceptor activity of tRNA and activities aminacyl-tRNA synthetases. After 20-min anoxic perfusion with the buffer containing Polyscias filicifolia Bailey biomass tincture, the acceptor activities of tRNA increased to the control value and activities of aminacyl-tRNA synthetases reached the control value. The acceptor activity of tRNA from isolated pig heart decreased and activities of aminacyl-tRNA synthetases increased under anoxia. Perfusion with buffer containing tincture of Polyscias filicifolia Bailey biomass restored acceptor activities of tRNA and activities of aminacyl-tRNA synthetases.

  18. Interferon titer and the 2',5'-oligoadenylate-synthetase activity in rat thymus lymphocytes in conditions of Omeprazol-caused hypergastrinemia

    Directory of Open Access Journals (Sweden)

    Kompanets I. V.

    2013-01-01

    Full Text Available The aim of this work was the determination of rat thymocytes response to hypergastrinemia evoked by hypoacidity and multiprobiotic «Symbiter® acidophilic concentrated» (symbiter treatment via the estimation of the interferon (IFN titer and 2', 5'-oligoadenylate (OA-synthetase activity in lymphocytes. 2', 5'-OA-synthetase is the IFN-induced enzyme. Methods. The micromethod of IFN titer determination by antiviral activity, spectrophotometrical method of 2', 5'-OA-synthetase activity determination. Results. It was shown that the IFN production by cultivated thymocytes is amplified while the 2', 5'-OA-synthetase activity decreases in these cells in conditions of hypoacidity caused by the 28-days omeprazol treatment. The treatment of animals by symbiter against a background of hypoacidity causes the augmentation of IFN production by thymocytes, but does not stimulate the 2', 5'-OA-synthetase activity. The IFN production by thymocytes in response to IFN inducers (PHA and cycloferone in vitro is intensified comparatively to the control at hypoacidity and symbiter treatment. Conclusions. The multiprobiotic symbiter exhibits interferonogenic properties. The IFN synthesis in response to induction in vitro is intensified in comparison with healthy animals at both hypoacidity and symbiter treatment while the 2', 5'-OA-synthetase acivity in thymocytes decreases.

  19. 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*

    Science.gov (United States)

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

    2015-01-01

    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. PMID:25422319

  20. 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.

    Science.gov (United States)

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Investigating the mechanism of ADP-forming acetyl-CoA synthetase from the protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Jones, Cheryl P; Khan, Kirin; Ingram-Smith, Cheryl

    2017-02-01

    ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II. © 2017 Federation of European Biochemical Societies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    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.

  3. Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model.

    Science.gov (United States)

    Shiue, Shih-Chang; Huang, Miao-Zeng; Tsai, Ting-Fen; Chang, Alice Chien; Choo, Kong Bung; Huang, Chiu-Jung; Su, Tsung-Sheng

    2015-01-23

    Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter. We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx

  4. Investigation of protein-ligand and protein-protein interactions in type II non-ribosomal peptide synthetases

    OpenAIRE

    Jaremko, Matt J.

    2017-01-01

    Non-ribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of many pharmaceutically relavant compounds. Type II NRPSs are an emerging subfamily of NRPSs that form hybrid pathways with type I fatty acid synthases (FAS), polyketide synthases (PKS), type I NRPSs, or others. The type II NRPSs commonly contain tailoring enzymes that generate unique substrate modifications, such as dehydrogenations and halogenation. Unlike type I NRPSs, the type II systems consists of standalone...

  5. A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8.

    Science.gov (United States)

    Kamada, Saho; Okugochi, Takahiro; Asano, Kaori; Tobe, Ryuta; Mihara, Hisaaki; Nemoto, Michiko; Inagaki, Kenji; Tamura, Takashi

    2016-10-01

    Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively.

  6. Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus1

    Science.gov (United States)

    Hlousek-Radojcic, Alenka; Evenson, Kimberly J.; Jaworski, Jan G.; Post-Beittenmiller, Dusty

    1998-01-01

    In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

  7. Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase*

    OpenAIRE

    Chen, Qingfeng; Westfall, Corey S.; Hicks, Leslie M.; Wang, Shiping; Jez, Joseph M.

    2010-01-01

    The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemi...

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

    Science.gov (United States)

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

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Fusion of the subunits α and β of succinyl-CoA synthetase as a phylogenetic marker for Pezizomycotina fungi

    Directory of Open Access Journals (Sweden)

    Amanda M. Koire

    2011-01-01

    Full Text Available Gene fusions, yielding the formation of multidomain proteins, are evolutionary events that can be utilized as phylogenetic markers. Here we describe a fusion gene comprising the α and β subunits of succinyl-coA synthetase, an enzyme of the TCA cycle, in Pezizomycotina fungi. This fusion is present in all Pezizomycotina with complete genome sequences and absent from all other organisms. Phylogenetic analysis of the α and β subunits of succinyl-CoA synthetase suggests that both subunits were duplicated and retained in Pezizomycotina while one copy was lost from other fungi. One of the duplicated copies was then fused in Pezizomycotina. Our results suggest that the fusion of the α and β subunits of succinyl-CoA synthetase can be used as a molecular marker for membership in the Pezizomycotina subphylum. If a species has the fusion it can be reliably classified as Pezizomycotina, while the absence of the fusion is suggestive that the species is not a member of this subphylum.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Rongzhong Li

    2015-07-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  13. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  14. Dexamethasone enhances glutamine synthetase activity and reduces N-methyl-D-aspartate neurotoxicity in mixed cultures of neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Edith Debroas

    2015-05-01

    Full Text Available Astrocytes are claimed to protect neurons against excitotoxicity by clearing glutamate from the extracellular space and rapidly converting it into glutamine. Glutamine, is then released into the extracellular medium, taken up by neurons and transformed back into glutamate which is then stored into synaptic vesicles. Glutamine synthetase (GS, the key enzyme that governs this glutamate/glutamine cycle, is known to be upregulated by glucocorticoids. In the present work we have thus studied in parallel the effects of dexamethasone on glutamine synthetase activity and NMDA-induced neuronal death in cultures derived from the brain cortex of murine embryos. We showed that dexamethasone was able to markedly enhance GS activity in cultures of astrocytes but not in near pure neuronal cultures. The pharmacological characteristics of the dexamethasone action strongly suggest that it corresponds to a typical receptor-mediated effect. We also observed that long lasting incubation (72 h of mixed astrocyte-neuron cultures in the presence of 100 nM dexamethasone significantly reduced the toxicity of NMDA treatment. Furthermore we demonstrated that methionine sulfoximine, a selective inhibitor of GS, abolished the dexamethasone-induced increase in GS activity and also markedly potentiated NMDA toxicity. Altogether these results suggest that dexamethasone may promote neuroprotection through a stimulation of astrocyte glutamine synthetase.

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

    Energy Technology Data Exchange (ETDEWEB)

    Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation); Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S., E-mail: esipov@mx.ibch.ru [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation)

    2017-01-15

    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°Ð¡, 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. P2{sub 1} 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.

  16. Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Matthias Ruwe

    2017-08-01

    Full Text Available The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (pppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP*Cg and RelSCg, exhibit high similarities to the conserved (pppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH protein RelCg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelSCg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelSCg was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP*Cg, as well as the slightly differing variant RelPCg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelSCg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important

  17. Mutations in PRPS1, which encodes the phosphoribosyl pyrophosphate synthetase enzyme critical for nucleotide biosynthesis, cause hereditary peripheral neuropathy with hearing loss and optic neuropathy (cmtx5).

    Science.gov (United States)

    Kim, Hee-Jin; Sohn, Kwang-Min; Shy, Michael E; Krajewski, Karen M; Hwang, Miok; Park, June-Hee; Jang, Sue-Yon; Won, Hong-Hee; Choi, Byung-Ok; Hong, Sung Hwa; Kim, Byoung-Joon; Suh, Yeon-Lim; Ki, Chang-Seok; Lee, Soo-Youn; Kim, Sun-Hee; Kim, Jong-Won

    2007-09-01

    We have identified missense mutations at conserved amino acids in the PRPS1 gene on Xq22.3 in two families with a syndromic form of inherited peripheral neuropathy, one of Asian and one of European descent. The disease is inherited in an X-linked recessive manner, and the affected male patients invariably develop sensorineural hearing loss of prelingual type followed by gating disturbance and visual loss. The family of European descent was reported in 1967 as having Rosenberg-Chutorian syndrome, and recently a Korean family with the same symptom triad was identified with a novel disease locus CMTX5 on the chromosome band Xq21.32-q24. PRPS1 (phosphoribosyl pyrophosphate synthetase 1) is an isoform of the PRPS gene family and is ubiquitously expressed in human tissues, including cochlea. The enzyme mediates the biochemical step critical for purine metabolism and nucleotide biosynthesis. The mutations identified were E43D, in patients with Rosenberg-Chutorian syndrome, and M115T, in the Korean patients with CMTX5. We also showed decreased enzyme activity in patients with M115T. PRPS1 is the first CMT gene that encodes a metabolic enzyme, shedding a new light on the understanding of peripheral nerve-specific metabolism and also suggesting the potential of PRPS1 as a target for drugs in prevention and treatment of peripheral neuropathy by antimetabolite therapy.

  18. Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia--MLASA syndrome.

    Science.gov (United States)

    Riley, Lisa G; Cooper, Sandra; Hickey, Peter; Rudinger-Thirion, Joëlle; McKenzie, Matthew; Compton, Alison; Lim, Sze Chern; Thorburn, David; Ryan, Michael T; Giegé, Richard; Bahlo, Melanie; Christodoulou, John

    2010-07-09

    Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA. Copyright 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  19. Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island.

    Science.gov (United States)

    Hadchouel, Alice; Wieland, Thomas; Griese, Matthias; Baruffini, Enrico; Lorenz-Depiereux, Bettina; Enaud, Laurent; Graf, Elisabeth; Dubus, Jean Christophe; Halioui-Louhaichi, Sonia; Coulomb, Aurore; Delacourt, Christophe; Eckstein, Gertrud; Zarbock, Ralf; Schwarzmayr, Thomas; Cartault, François; Meitinger, Thomas; Lodi, Tiziana; de Blic, Jacques; Strom, Tim M

    2015-05-07

    Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  20. Structural and Functional Characterization of Aerobactin Synthetase IucA from a Hypervirulent Pathotype of Klebsiella pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Daniel C.; Drake, Eric J.; Grant, Thomas D.; Gulick, Andrew M. (Buffalo); (HWMRI)

    2016-06-28

    Iron is a vital mineral nutrient required by virtually all life forms to prosper; pathogenic bacteria are no exception. Despite the abundance of iron within the human host, highly regulated iron physiology can result in exceedingly low levels of iron bioavailable to prospective invading bacteria. To combat this scarcity of iron, many pathogenic bacteria have acquired specific and efficient iron acquisition systems, which allow them to thrive in iron-deficient host environments. One of the more prominent bacterial iron acquisition systems involves the synthesis, secretion, and reuptake of small-molecule iron chelators known as siderophores. Aerobactin, a citrate-hydroxamate siderophore originally isolated nearly 50 years ago, is produced by a number of pathogenic Gram-negative bacteria. Aerobactin has recently been demonstrated to play a pivotal role in mediating the enhanced virulence of a particularly invasive pathotype of Klebsiella pneumoniae (hvKP). Toward further understanding of this key virulence factor, we report the structural and functional characterization of aerobactin synthetase IucA from a strain of hvKP. The X-ray crystal structures of unliganded and ATP-bound forms of IucA were solved, forming the foundation of our structural analysis. Small angle X-ray scattering (SAXS) data suggest that, unlike its closest structurally characterized homologues, IucA adopts a tetrameric assembly in solution. Finally, we employed activity assays to investigate the substrate specificity and determine the apparent steady-state kinetic parameters of IucA.

  1. Regulation of hepatocyte-specific gene expression in cultures of human embryonic hepatocytes

    NARCIS (Netherlands)

    van Roon, M. A.; Zonneveld, D.; de Boer, P. A.; Moorman, A. F.; Charles, R.; Lamers, W. H.

    1990-01-01

    The aim of this study was to see whether the rat embryo can serve as a model system for hepatocyte-specific gene expression in the human embryo. Carbamoylphosphate synthetase was used as a hepatocyte-specific marker molecule. Despite the earlier developmental appearance of this enzyme in human than

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

    Science.gov (United States)

    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. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Cannabidiol protects retinal neurons by preserving glutamine synthetase activity in diabetes

    Science.gov (United States)

    El-Remessy, A.B.; Khalifa, Y.; Ibrahim, A.S.; Liou, G.I.

    2010-01-01

    Purpose We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process. Methods Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, Müller cell activation, GS tyrosine nitration, and GS activity were determined. Results Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with Müller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats. Conclusions These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration. PMID:20806080

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

    Science.gov (United States)

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

    2017-04-01

    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. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

    Directory of Open Access Journals (Sweden)

    Niran Roongsawang

    2010-12-01

    Full Text Available Lipopeptide biosurfactants (LPBSs consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive and Pseudomonas (Gram-negative have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs. Production of active‑form NRPSs requires not only transcriptional induction and translation but also post‑translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

  6. Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

    Science.gov (United States)

    Amaya, Kensey R; Kocherginskaya, Svetlana A; Mackie, Roderick I; Cann, Isaac K O

    2005-11-01

    Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the gamma-glutamyl transferase (gamma-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.

  7. Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase.

    Science.gov (United States)

    Oliver, Justin C; Gudihal, Ravidra; Burgner, John W; Pedley, Anthony M; Zwierko, Alexander T; Davisson, V Jo; Linger, Rebecca S

    2014-03-01

    GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Selenophosphate synthetase in the male accessory glands of an insect without selenoproteins.

    Science.gov (United States)

    Fuessl, Marion; Reinders, Jörg; Oefner, Peter J; Heinze, Jürgen; Schrempf, Alexandra

    2014-12-01

    Selenoproteins (containing the 21st proteinogenic amino acid selenocysteine) play important roles throughout all domains of life. Surprisingly, a number of taxa have small selenoproteomes, and Hymenopteran insects appear to have fully lost selenoproteins. Nevertheless, their genomes contain genes for several proteins of the selenocysteine insertion machinery, including selenophosphate synthetase 1 (SELD/SPS1). At present, it is unknown whether this enzyme has a selenoprotein-independent function, and whether the gene is actually translated into a protein in Hymenoptera. Here, we report that SELD/SPS1 is present as a protein in the accessory glands of males of the ant Cardiocondyla obscurior. It appears to be more abundant in the glands of winged disperser males than in those of wingless, local fighter males. Mating increases the lifespan and fecundity of queens in C. obscurior, and mating with winged males has a stronger effect on queen fitness than mating with a wingless male. SELD/SPS 1 has been suggested to play an important role in oxidative stress defense, and might therefore be involved in the life-prolonging effect of mating. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Margarita eGarcía-Calderón

    2015-09-01

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

  10. Dynamic QTL analysis of protein content and glutamine synthetase activity in recombinant inbred wheat lines.

    Science.gov (United States)

    Li, H M; Liang, H; Li, Z; Tang, Z X; Fu, S L; Geng, Y Y; Yan, B J; Ren, Z L

    2015-07-31

    Protein content (PC) is a crucial factor that determines the end-use and nutritional quality of wheat (Triticum aestivum). Glutamine synthetase (GS), which is a major participant in nitrogen metabolism, can convert inorganic nitrogen into organic nitrogen. Although many studies have been conducted on PC and GS, a dynamic analysis of all of the filling stages has not been conducted. Therefore, 115 F9-10 recombinant inbred wheat lines of 'R131/R142' were used to analyze PC and GS activity during different developmental stages, using the conditional quantitative trait loci (QTL) mapping method. Twenty-two and six conditional QTL were detected for PC and GS activily, respectively. More QTL in leaf PC were detected during the early filling stages than in the later filling stages. Grain PC QTL displayed different dynamic variations to leaf PC QTL during the entire grain-filling stages. All of the QTL were expressed differently over time, and nine conditional QTL were detected across two filling stages. QTL with similar functions may have tended to group in specific locales. This study provides dynamic genetic information on protein accumulation during grain-filling stages.

  11. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera

    DEFF Research Database (Denmark)

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just

    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...

  12. Expression of asparagine synthetase genes in sunflower (Helianthus annuus) under various environmental stresses.

    Science.gov (United States)

    Herrera-Rodríguez, María Begoña; Pérez-Vicente, Rafael; Maldonado, José-María

    2007-01-01

    In sunflower, asparagine synthetase (AS; EC 6.3.5.4) is encoded by a small family of three genes (HAS1, HAS1.1 and HAS2) that are differentially regulated by light, carbon and nitrogen availability. In this study, the response of each gene to various stress conditions was examined by Northern analysis with gene-specific probes in leaves and roots. The expression of HAS1 and HAS1.1 genes was induced by osmotic stress (300 mM mannitol), salt stress (150 mM NaCl), and heavy-metal stress (20 microM CuSO(4)), more in roots than in leaves. The expression of HAS2 was not significantly altered by stress treatments. The positive response of HAS1 and HAS1.1 genes to osmotic and salt stresses occurred in the light, in contrast to that previously found in unstressed plants. Measurements of sucrose and total free amino acid contents in leaves and roots indicate that the expression of root HAS1 and HAS1.1 genes in stressed plants is not under metabolic control by the intracellular C/N ratio, suggesting the involvement of some specific stress factor(s). Growth of plants at 40 degrees C for 12h negatively affected the expression of HAS1 and HAS1.1 but not that of HAS2.

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

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru; Abramchik, Yu. A., E-mail: tostars@mail.ru; Zhukhlistova, N. E., E-mail: ugama@yandex.ru; Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-09-15

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

  14. Nuclear glutamine synthetase evolution in Nicotiana: phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids.

    Science.gov (United States)

    Clarkson, James J; Kelly, Laura J; Leitch, Andrew R; Knapp, Sandra; Chase, Mark W

    2010-04-01

    Interspecies relationships in Nicotiana (Solanaceae) are complex because 40 species are diploid (two sets of chromosomes) and 35 species are allotetraploid (four sets of chromosomes, two from each progenitor diploid species). We sequenced a fragment (containing four introns) of the nuclear gene 'chloroplast-expressed glutamine synthetase' (ncpGS) in 65 species of Nicotiana. Here we present the first phylogenetic analysis based on a low-copy nuclear gene for this well studied and important genus. Diploid species have a single-copy of ncpGS, and allotetraploids as expected have two homeologous copies, each derived from their progenitor diploid. Results were particularly useful for determining the paternal lineage of previously enigmatic taxa (for which our previous analyses had revealed only the maternal progenitors). In particular, we were able to shed light on the origins of the two oldest and largest allotetraploid sections, N. sects. Suaveolentes and Repandae. All homeologues have an intact reading frame and apparently similar rates of divergence, suggesting both remain functional. Difficulties in fitting certain diploid species into the sectional classification of Nicotiana on morphological grounds, coupled with discordance between the ncpGS data and previous trees (i.e. plastid, nuclear ribosomal DNA), indicate a number of homoploid (diploid) hybrids in the genus. We have evidence for Nicotiana glutinosa and Nicotiana linearis being of hybrid origin and patterns of intra-allelic recombination also indicate the possibility of reticulate origins for other diploid species. (c) 2009 Elsevier Inc. All rights reserved.

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

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    Park Jin

    2011-08-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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.

  17. Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

    KAUST Repository

    Hohl, Adrian

    2017-12-06

    The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

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

    Science.gov (United States)

    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.

  19. Effect of bromochloromethane and fumarate on phylogenetic diversity of the formyltetrahydrofolate synthetase gene in bovine rumen.

    Science.gov (United States)

    Mitsumori, Makoto; Matsui, Hiroki; Tajima, Kiyoshi; Shinkai, Takumi; Takenaka, Akio; Denman, Stuart E; McSweeney, Christopher S

    2014-01-01

    Effect of the methane inhibitor, bromochloromethane (BCM) and dietary substrate, fumarate, on microbial community structure of acetogen bacteria in the bovine rumen was investigated through analysis of the formyltetrahydrofolate synthetase gene (fhs). The fhs sequences obtained from BCM-untreated, BCM-treated, fumarate-untreated and fumarate-treated bovine rumen were categorized into homoacetogens and nonhomoacetogenic bacteria by homoacetogen similarity scores. Phylogenetic tree analysis indicated that most of the fhs sequences categorized into homoacetogens were divided into nine clusters, which were in close agreement with a result shown in a self-organizing map. The diversity of the fhs sequences from the BCM-treated rumen was significantly different from those from BCM-non-treated rumen. Principal component analysis also showed that addition of BCM to the rumen altered the population structure of acetogenic bacteria significantly but the effect of fumarate was comparatively minor. These results indicate that BCM affects diversity of actogens in the bovine rumen, and changes in acetogenic community structure in response to methane inhibitors may be caused by different mechanisms. © 2013 Japanese Society of Animal Science.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-10-07

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

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

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available 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.

  3. Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

    Directory of Open Access Journals (Sweden)

    Drasko Boko

    2010-01-01

    Full Text Available Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS, evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. [Nitric oxide synthetase and carbon monoxide decrease in the penile corpus cavernous of hyper homocysteine rats].

    Science.gov (United States)

    Chen, Qing-Jun; Cao, Hui-Feng; Zhang, Dong-Sheng; Yang, Yu-Qi; Qin, Wen-Bo; Hu, Cun-Li; Hao, Peng

    2008-08-01

    To detect the levels of nitric oxide synthetase (NOS) and carbon monoxide (CO) in the penile corpus cavernous of adult male Wistar rats with high homocysteine (Hhcy) and to explore the relationship of NOS and CO levels with erectile dysfunction. Twenty Wistar rats were equally and randomly divided into a control and an Hhcy group and fed on normal diet and normal diet with 3.0% methionine respectively. Four weeks later, the levels of NOS and CO in the penile corpus cavernous were detected by ultraviolet spectrophotometry and that of serum homocysteine by the cycle enzyme method. Compared with the control group, the levels of NOS and CO in the penile corpus cavernous were significantly lower in the Hhcy group, (6.45 +/- 1.12) nmol/(g x min) vs (10.77 +/- 0.60) nmol/(g x min) and (10.60 +/- 0.92) micromol/L vs (13.36 +/- 0.44) micromol/L, while that of homocysteine was significantly higher, (22.32 +/- 1.65) micromol/L) vs (4.90 +/- 1.73) micromol/L. Four-week diet with methionine can cause Hhcy and significantly decreased levels of NOS and CO in the penile corpus cavernous in Wistar rats. Hhcy is an independent risk factor of erectile dysfunction.

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

    Science.gov (United States)

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

    1997-01-01

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

  7. Functional identification of glutamate cysteine ligase and glutathione synthetase in the marine yeast Rhodosporidium diobovatum

    Science.gov (United States)

    Kong, Min; Wang, Fengjuan; Tian, Liuying; Tang, Hui; Zhang, Liping

    2018-02-01

    Glutathione (GSH) fulfills a variety of metabolic functions, participates in oxidative stress response, and defends against toxic actions of heavy metals and xenobiotics. In this study, GSH was detected in Rhodosporidium diobovatum by high-performance liquid chromatography (HPLC). Then, two novel enzymes from R. diobovatum were characterized that convert glutamate, cysteine, and glycine into GSH. Based on reverse transcription PCR, we obtained the glutathione synthetase gene ( GSH2), 1866 bp, coding for a 56.6-kDa protein, and the glutamate cysteine ligase gene ( GSH1), 2469 bp, coding for a 90.5-kDa protein. The role of GSH1 and GSH2 for the biosynthesis of GSH in the marine yeast R. diobovatum was determined by deletions using the CRISPR-Cas9 nuclease system and enzymatic activity. These results also showed that GSH1 and GSH2 were involved in the production of GSH and are thus being potentially useful to engineer GSH pathways. Alternatively, pET- GSH constructed using vitro recombination could be used to detect the function of genes related to GSH biosynthesis. Finally, the fermentation parameters determined in the present study provide a reference for industrial GSH production in R. diobovatum.

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

    Directory of Open Access Journals (Sweden)

    Massimo Ferrara

    2013-04-01

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

  9. Role of tRNAPro in pretransfer editing of alanine by prolyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Boyarshin K. S.

    2013-09-01

    Full Text Available Aim. To characterize the process of tRNA-dependent pretransfer edi- ting of alanine by prolyl-tRNA synthetase of bacteria Enterococcus faecalis (ProRSEf. Methods. Velocity of the editing processes in vitro was determined by ATP hydrolysis by ProRSEf. Pretransfer and posttransfer editing were experimentally separated by site-directed mutagenesis. Results. tRNA-dependent pretransfer editing is characterized by three-fold larger velocity then tRNA-independent editing. Effectivity of the process depends on the presence of 2'-hydroxyle group of A76 tRNAPro. In the absence of tRNAPro selective release of alanyl-AMP occurs simultaneously with tRNA-independent pretransfer editing. Released alanyl-AMP can be re-bound and hydrolyzed. Conclusions. tRNA-dependent pretransfer editing of alanine by ProRSEf is the catalytic mechanism, mediated by 2'-hydroxyl group of A76 tRNAPro. In the absence of tRNAPro tRNA-independent pretransfer editing and selective release of alanyl-AMP occur.

  10. Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps.

    Science.gov (United States)

    Cvetesic, Nevena; Perona, John J; Gruic-Sovulj, Ita

    2012-07-20

    Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.

  11. Kinetic Partitioning between Synthetic and Editing Pathways in Class I Aminoacyl-tRNA Synthetases Occurs at Both Pre-transfer and Post-transfer Hydrolytic Steps*

    Science.gov (United States)

    Cvetesic, Nevena; Perona, John J.; Gruic-Sovulj, Ita

    2012-01-01

    Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps. PMID:22648413

  12. Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Eriko Kage-Nakadai

    Full Text Available In multicellular organisms, the surface barrier is essential for maintaining the internal environment. In mammals, the barrier is the stratum corneum. Fatty acid transport protein 4 (FATP4 is a key factor involved in forming the stratum corneum barrier. Mice lacking Fatp4 display early neonatal lethality with features such as tight, thick, and shiny skin, and a defective skin barrier. These symptoms are strikingly similar to those of a human skin disease called restrictive dermopathy. FATP4 is a member of the FATP family that possesses acyl-CoA synthetase activity for very long chain fatty acids. How Fatp4 contributes to skin barrier function, however, remains to be elucidated. In the present study, we characterized two Caenorhabditis elegans genes, acs-20 and acs-22, that are homologous to mammalian FATPs. Animals with mutant acs-20 exhibited defects in the cuticle barrier, which normally prevents the penetration of small molecules. acs-20 mutant animals also exhibited abnormalities in the cuticle structure, but not in epidermal cell fate or cell integrity. The acs-22 mutants rarely showed a barrier defect, whereas acs-20;acs-22 double mutants had severely disrupted barrier function. Moreover, the barrier defects of acs-20 and acs-20;acs-22 mutants were rescued by acs-20, acs-22, or human Fatp4 transgenes. We further demonstrated that the incorporation of exogenous very long chain fatty acids into sphingomyelin was reduced in acs-20 and acs-22 mutants. These findings indicate that C. elegans Fatp4 homologue(s have a crucial role in the surface barrier function and this model might be useful for studying the fundamental molecular mechanisms underlying human skin barrier and relevant diseases.

  13. Germ cell differentiation-dependent and stage-specific expression of LANCL1 in rodent testis

    DEFF Research Database (Denmark)

    Nielsen, J E; Hansen, Martin Asser; Jørgensen, Marianne

    2003-01-01

    LANCL1 (LanC-like protein 1) is related to the bacterial LanC (lanthionine synthetase C) family, which is involved in the biosynthesis of antimicrobial peptides. Highest expression levels of LANCL1 are found in testes and brain, two organs that exist behind blood-tissue barriers. In the mouse, th...

  14. Hydrogenosomal succinyl-CoA synthetase from the rumen-dwelling fungus Neocallimastix patriciarum; an energy-producing enzyme of mitochondrial origin.

    Science.gov (United States)

    Dacks, Joel B; Dyal, Patricia L; Embley, T Martin; van der Giezen, Mark

    2006-05-24

    Hydrogenosomes are hydrogen-producing organelles that are related to mitochondria and found in a variety of evolutionarily unrelated anaerobic microbial eukaryotes. Similar to classic mitochondria, hydrogenosomes contain the enzyme catalyzing the only reaction of the citric acid cycle directly producing energy; succinyl-CoA synthetase. We have isolated and characterized the genes encoding both subunits of this enzyme from the anaerobic chytrid fungus Neocallimastix patriciarum, a model organism in hydrogenosome research. Both subunits contain all characteristic features of this enzyme, including predicted hydrogenosomal targeting signals. Phylogenetic analyses of succinyl-CoA synthetase clearly indicate its mitochondrial ancestry, both by affiliation with mitochondrially localized fungal homologues and by the sisterhood of the eukaryotic succinyl-CoA synthetase clade with alpha-proteobacteria. Our analyses of the Trichomonas vaginalis SCS sequences also confirmed the mitochondrial affiliation of these hydrogenosomal enzymes, in contrast to previous results. While both hydrogenosomal and mitochondrial succinyl-CoA synthetase homologues have been identified, no succinyl-CoA synthetase proteins were identifiable in taxa possessing another mitochondrially derived organelle, the mitosome. Our analyses further confirm the mitochondrial ancestry of the Neocallimastix hydrogenosome and sheds light upon the stepwise process by which mitochondria evolve into alternate forms of the organelle.

  15. Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules 1

    Science.gov (United States)

    Groat, R. Gene; Schrader, Larry E.

    1982-01-01

    Host plant glutamine synthetase (GS) has been purified 100-fold from N2-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody-containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N2-fixing alfalfa nodules. Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts. Images Fig. 2 Fig. 3 Fig. 4 PMID:16662757

  16. Expression and specific activities of carbamoyl phosphate synthetase 1 in chronic hypoxic rats

    Directory of Open Access Journals (Sweden)

    Uly A. Nikmah

    2016-04-01

    Full Text Available Background: Urea biosynthesis is a very important process in the liver which needs ATP, CO2 and functional mitochondria or aerobic condition. Liver can adapt to hypoxic condition, generally and locally. This study aimed to analyze the effect of chronic hypoxia on liver urea biosynthesis as indicated by the level and specific activity of mRNA of carbamoyl phosphate synthetase 1 (CPS1, a key enzyme in urea biosynthesis in hypoxic rats.Methods: 20 male Sprague-Dawley rats were placed in hypoxic chamber supplied by a mixture of 10% O2 and 90% N2. Five rats were sacrificed at 1, 3, 5, and 7 days after exposure. Liver homogenates were analyzed for HIF-1 (hypoxia inducible factor-1 by ELISA, CPS1 mRNA by real time RT-PCR and CPS1 enzymatic specific activities by Pierson method. Data were analyzed by ANOVA test and Pearson correlation.Results: The HIF-1 in liver increased significantly, as well as CPS1 mRNA and CPS1 enzymatic activities (p<0.05. There was a strong correlation (r=0.618; p<0.01 between the level of CPS1 mRNA and CPS1 enzymatic activities, moderate correlation between HIF-1 and CPS1 mRNA (r=0.419; p<0.05 but no correlation between HIF-1 and CPS1 enzymatic activities. The study indicated that urea biosynthesis in liver was affected by hypoxia and partially under HIF-1 regulation. The study also found increase of urea and NH3 biosynthesis related to proteolysis as indicated by the decrease of total body weight and liver weight.Conclusion: There was an increase in the expression and specific activities of CPS1 in urea biosynthesis as a result of increasing proteolysis  in chronic hypoxic condition.

  17. Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

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    Benítez, Diego; Medeiros, Andrea; Fiestas, Lucía; Panozzo-Zenere, Esteban A; Maiwald, Franziska; Prousis, Kyriakos C; Roussaki, Marina; Calogeropoulou, Theodora; Detsi, Anastasia; Jaeger, Timo; Šarlauskas, Jonas; Peterlin Mašič, Lucíja; Kunick, Conrad; Labadie, Guillermo R; Flohé, Leopold; Comini, Marcelo A

    2016-04-01

    The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad

  18. Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

    Directory of Open Access Journals (Sweden)

    Diego Benítez

    2016-04-01

    Full Text Available The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS, catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate.A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively, and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl diamine derivatives and an N5-substituted paullone (MOL2008 halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range and Leishmania infantum promastigotes (EC50 = 12 μM, respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds.Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent

  19. Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

    Science.gov (United States)

    Mariotti, Marco; Santesmasses, Didac; Capella-Gutierrez, Salvador; Mateo, Andrea; Arnan, Carme; Johnson, Rory; D'Aniello, Salvatore; Yim, Sun Hee; Gladyshev, Vadim N; Serras, Florenci; Corominas, Montserrat; Gabaldón, Toni; Guigó, Roderic

    2015-09-01

    Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome. © 2015 Mariotti et al.; Published by Cold Spring Harbor Laboratory Press.

  20. Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism.

    Science.gov (United States)

    Wang, Yi; Chang, Le; Zhai, Jiahui; Wu, Qiao; Wang, Donggen; Wang, Yunfang

    2017-12-01

    Both primary hepatocytes and stem cells-derived hepatocyte-like cells (HLCs) are major sources for bioartificial liver (BAL). Maintenance of hepatocellular functions and induction of functional maturity of HLCs are critical for BAL's support effect. It remains difficult to assess and improve detoxification functions inherent to hepatocytes, including ammonia clearance. Here, we aim to assess ammonia metabolism and identify ammonia detoxification enhancer by developing an imaging strategy. In hepatoma cell line HepG2, and immortalized hepatic cell line LO2, carbamoyl phosphate synthetase 1 (CPS1) gene, the first enzyme of ammonia-eliminating urea cycle, was labelled with fluorescence protein via CRISPR/Cas9 system. With the reporter-based screening approach, cellular detoxification enhancers were selected among a collection of 182 small molecules. In both CPS1 reporter cell lines, the fluorescence intensity is positively correlated with cellular CPS1 mRNA expression, ammonia elimination and secreted urea, and reflected ammonia detoxification in a dose-dependent manner. Surprisingly, high-level CPS1 reporter clones also reserved many other critical hepatocellular functions, for example albumin secretion and cytochrome 450 metabolic functions. Sodium phenylbutyrate and resveratrol were identified to enhance metabolism-related gene expression and liver-enriched transcription factors C/EBPα, HNF4α. In conclusion, the CPS1-reporter system provides an economic and effective platform for assessment of cellular metabolic function and high-throughput identification of chemical compounds that improve detoxification activities in hepatic lineage cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  1. Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio.

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    Schaper, Wiebke; Bentrop, Joachim; Ustinova, Jana; Blume, Linda; Kats, Elina; Tiralongo, Joe; Weinhold, Birgit; Bastmeyer, Martin; Münster-Kühnel, Anja-K

    2012-04-13

    Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

  2. Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

    Science.gov (United States)

    Di, Wu; Fujita, Akiko; Hamaguchi, Kayo; Delannoy, Philippe; Sato, Chihiro; Kitajima, Ken

    2017-04-01

    The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Functional role of C-terminal domain of Thermus thermophilus leucyl-tRNA synthetase

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    Tukalo M. A.

    2010-11-01

    Full Text Available Aim. To study a role of C-terminal domain of T. thermophilus leucyl-tRNA synthetase (LeuRSTT in the reactions of aminoacylation and editing. Methods. A mutant of LeuRSTT without C- terminal domain (ΔС was obtained by the method of mutagenesis. The kinetic constants in aminoacylation reaction catalyzed by LeuRS and its mutant (ΔС were determined by the methods of equilibrium enzyme kinetics. To evaluate the contribution of C-terminal domain to interaction of the enzyme with tRNALeu, Kd of a complex between tRNA and LeuRSTT and its mutant ΔС was determined by fluorescence titration. Results. The C-terminal domain is shown to play a significant role in the aminoacylation and editing reactions of LeuRSTT and not essential for the activity in the reaction of amino acid activation. The kinetic parameters of aminoacylation of tRNALeu and tRNATyr by LeuRS and ΔС mutant were also determined, their analysis suggests that the C-domain is not critical for the manifestation of specificity of the enzyme in the recognition of homologous RNAs. At the same time a significant influence of the C-terminal domain on the value of catalytic constant was shown. At the domain deletion the kcat value is lower by 152-fold. Conclusion. The C-terminal domain of LeuRSTT is evolutionarily acquired to enhance the rate of catalysis in the aminoacylation and editing reactions, and makes no significant contribution to the specificity of the enzyme in the recognition of tRNA.

  4. Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc.

    Science.gov (United States)

    Genuário, Diego Bonaldo; Silva-Stenico, Maria Estela; Welker, Martin; Beraldo Moraes, Luiz Alberto; Fiore, Marli Fátima

    2010-04-01

    A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, São Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 16S rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 16S rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin. Copyright 2009 Elsevier Ltd. All rights reserved.

  5. A mathematical model for the adenylosuccinate synthetase reaction involved in purine biosynthesis

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    Likhoshvai Vitalii A

    2007-02-01

    Full Text Available Abstract Background Development of the mathematical models that adequately describe biochemical reactions and molecular-genetic mechanisms is one of the most important tasks in modern bioinformatics. Because the enzyme adenylosuccinate synthetase (AdSS has long been extensively studied, a wealth of kinetic data has been accumulated. Results We describe a mathematical model for the reaction catalyzed by AdSS. The model's parameters were fitted to experimental data obtained from published literature. The advantage of our model is that it includes relationships between the reaction rate, the concentrations of three substrates (GTP, IMP and ASP, the effects of five inhibitors (GMP, GDP, AMP, ASUC and SUCC, and the influence of Mg2+ ions. Conclusion Our model describes the reaction catalyzed by AdSS as a fully random process. The model structure implies that each of the inhibitors included in it is only competitive to one of the substrates. The model was tested for adequacy using experimental data published elsewhere. The values obtained for the parameters are as follows: Vmax = 1.35·10-3 mM/min, KmGTP = 0.023 mM, KmIMP = 0.02 mM, KmASP = 0.3 mM, KiGMP = 0.024 mM, KiGDP = 8·10-3 mM, KiAMP = 0.01 mM, KiASUC = 7.5·10-3 mM, KiSUCC = 8 mM, KmMg = 0.08 mM.

  6. Identification of Staphylococcus argenteus in Eastern China based on a nonribosomal peptide synthetase (NRPS) gene.

    Science.gov (United States)

    Zhang, Dao-Feng; Xu, Xuebin; Song, Qifa; Bai, Yalong; Zhang, Yi; Song, Minghui; Shi, Chunlei; Shi, Xianming

    2016-09-01

    To investigate whether the Staphylococcus argenteus is present in Eastern China and to verify the utility of a new screening process. Phenotype observation, PCR assay targeting a hypothetical nonribosomal peptide synthetase (NRPS) gene, phylogenetic analysis of rpoB and multilocus sequence typing were used to screen and identify strains of S. argenteus from 839 presumptive S. aureus isolates. Eighty-nine (89/839, 10.6%) of the presumptive S. aureus isolates produced white colonies on tryptone soya agar plates. Of the white-colony isolates, six (6/89, 7%) were S. argenteus, 75 (75/89, 84%) were S. aureus and eight (8/89, 9%) were other bacteria. The PCR-based method targeting the NRPS gene can simultaneously identify and distinguish S. argenteus and S. aureus. All representative sequences of rpoB generated in this study were deposited in GenBank under accession numbers SJTU F20002, KT767581; SJTU F20269, KT767582; SJTU F20419, KT767583; SJTU F20420, KT767584; SJTU F20124, KT767585; SJTU F21164, KT767586; SJTU F21285, KT767587; SJTU F21224, KT767588; SJTU F21155, KT767589; SJTU F21294, KT767590; SJTU F20030, KT767591; SJTU F20044, KT767592; SJTU F20135, KT767593; SJTU F20123, KT767594; SJTU F21319, KT767595, respectively. All the new sequence types (STs) were submitted to a multilocus sequence typing database and the assigned ST numbers are ST3261 (151-469-20-101-145-150-131), ST3262 (12-3-1-1-4-4-410) and ST3267 (2-471-2-2-6-3-2).

  7. A sensing role of the glutamine synthetase in the nitrogen regulation network in Fusarium fujikuroi.

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    Dominik Wagner

    Full Text Available In the plant pathogenic ascomycete Fusarium fujikuroi the synthesis of several economically important secondary metabolites (SM depends on the nitrogen status of the cells. Of these SMs, gibberellin and bikaverin synthesis is subject to nitrogen catabolite repression (NCR and is therefore only executed under nitrogen starvation conditions. How the signal of available nitrogen quantity and quality is sensed and transmitted to transcription factors is largely unknown. Earlier work revealed an essential regulatory role of the glutamine synthetase (GS in the nitrogen regulation network and secondary metabolism as its deletion resulted in total loss of SM gene expression. Here we present extensive gene regulation studies of the wild type, the Δgln1 mutant and complementation strains of the gln1 deletion mutant expressing heterologous GS-encoding genes of prokaryotic and eukaryotic origin or 14 different F. fujikuroi gln1 copies with site-directed mutations. All strains were grown under different nitrogen conditions and characterized regarding growth, expression of NCR-responsive genes and biosynthesis of SM. We provide evidence for distinct roles of the GS in sensing and transducing the signals to NCR-responsive genes. Three site directed mutations partially restored secondary metabolism and GS-dependent gene expression, but not glutamine formation, demonstrating for the first time that the catalytic and regulatory roles of GS can be separated. The distinct mutant phenotypes show that the GS (1 participates in NH4 (+-sensing and transducing the signal towards NCR-responsive transcription factors and their subsequent target genes; (2 affects carbon catabolism and (3 activates the expression of a distinct set of non-NCR GS-dependent genes. These novel insights into the regulatory role of the GS provide fascinating perspectives for elucidating regulatory roles of GS proteins of different organism in general.

  8. Proximal tubule glutamine synthetase expression is necessary for the normal response to dietary protein restriction.

    Science.gov (United States)

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

    2017-07-01

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

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

    Science.gov (United States)

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

    2013-10-01

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

  10. Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

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    Anna P Lucarelli

    Full Text Available The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB and extensively drug-resistant strains (XDR-TB is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+ or Mn(2+ and inhibited by Ca(2+ and Cu(2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+ and Cu(2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

  11. Interstitial lung disease in anti-synthetase syndrome: Initial and follow-up CT findings

    Energy Technology Data Exchange (ETDEWEB)

    Debray, Marie-Pierre, E-mail: marie-pierre.debray@bch.aphp.fr [AP-HP, Bichat-Claude Bernard Hospital, Department of Radiology, 46, rue Henri Huchard, 75877 Paris Cedex 18 (France); Borie, Raphael, E-mail: raphael.borie@bch.aphp.fr [AP-HP, Bichat-Claude Bernard Hospital, Department of Pneumology A and Centre de Compétence Maladies Pulmonaires rares, DHU Fire 46, rue Henri Huchard, 75877 Paris Cedex 18 (France); Inserm, U1152, Paris (France); Revel, Marie-Pierre, E-mail: marie-pierre.revel@htd.aphp.fr [AP-HP, Cochin Hospital, Department of Radiology, 27, Rue du Fg Saint Jacques, 75679 Paris Cedex 14 (France); Naccache, Jean-Marc, E-mail: jean-marc.naccache@tnn.aphp.fr [AP-HP, Avicenne Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, Bobigny (France); AP-HP, Tenon Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, 4, rue de la Chine, 75020 Paris (France); Khalil, Antoine, E-mail: antoine.khalil@tnn.aphp.fr [AP-HP, Tenon Hospital, Department of Radiology, 4, rue de la Chine, 75020 Paris (France); Toper, Cécile, E-mail: cecile.toper@gmail.com [AP-HP, Tenon Hospital, Department of Pneumology and Centre de Compétence Maladies Pulmonaires rares, 4, rue de la Chine, 75020 Paris (France); Israel-Biet, Dominique, E-mail: dominique.israel-biet@egp.aphp.fr [Université Paris Descartes and AP-HP, Department of Pneumology, Georges Pompidou European Hospital, 20, rue Leblanc, 75015 Paris (France); and others

    2015-03-15

    Purpose: To describe the initial and follow-up CT features of interstitial lung disease associated with anti-synthetase syndrome (AS-ILD). Materials and methods: Two independent thoracic radiologists retrospectively analysed thin-section CT images obtained at diagnosis of AS-ILD in 33 patients (17 positive for anti-Jo1, 13 for anti-PL12, and three for anti-PL7 antibodies). They evaluated the pattern, distribution and extent of the CT abnormalities. They also evaluated the change in findings during follow-up (median 27 months; range 13–167 months) in 26 patients. Results: At diagnosis, ground-glass opacities (100%), reticulations (87%) and traction bronchiectasis (76%) were the most common CT findings. Consolidations were present in 45% of patients. A non-specific interstitial pneumonia (NSIP), organizing pneumonia (OP) or mixed NSIP-OP CT pattern were observed in 15 out of 33 (45%), seven out of 33 (21%) and eight out of 33 (24%) patients, respectively, whereas the CT pattern was indeterminate in three patients. During follow-up, consolidations decreased or disappeared in 11 out of 12 patients (92%), among which seven within the first 6 months, but honeycombing progressed or appeared in ten out of 26 patients (38%) and overall disease extent increased in nine out of 26 patients (35%). Conclusion: CT features at diagnosis of AS-ILD mainly suggest NSIP and OP, isolated or in combination. Consolidations decrease or disappear in most cases but the disease may progress to fibrosis in more than one third of patients.

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

    Science.gov (United States)

    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.

  13. MS_RHII-RSD, a dual-function RNase HII-(p)ppGpp synthetase from Mycobacterium smegmatis.

    Science.gov (United States)

    Murdeshwar, Maya S; Chatterji, Dipankar

    2012-08-01

    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 Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] 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 actRel(Msm) 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.

  14. Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules.

    Science.gov (United States)

    Silvente, Sonia; Reddy, Pallavolu M; Khandual, Sanghamitra; Blanco, Lourdes; Alvarado-Affantranger, Xochitl; Sanchez, Federico; Lara-Flores, Miguel

    2008-01-01

    A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.

  15. A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

    Directory of Open Access Journals (Sweden)

    Nederbragt Alexander J

    2009-08-01

    Full Text Available Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS. Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454" and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully

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

    CSIR Research Space (South Africa)

    Theron, Anjo

    2017-10-01

    Full Text Available Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform A. Theron1, R. L. Roth1, H. Hoppe1¤, C. Parkinson2, C. W. van der Westhuyzen1, S. Stoychev1, I. Wiid3, R. D. Pietersen3, B... contributes directly to PLOS ONE | https://doi.org/10.1371/journal.pone.0185068 October 3, 2017 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPENACCESS Citation: Theron A, Roth RL, Hoppe H, Parkinson C, van der Westhuyzen CW, Stoychev S...

  17. The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

    Directory of Open Access Journals (Sweden)

    Nkosi Thokozani C

    2011-07-01

    Full Text Available Abstract Background The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the

  18. Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1.

    Science.gov (United States)

    Iwaki, Jun; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Hasegawa, Tsunemi

    2005-11-01

    Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 66.1, c = 196.2 A, and diffracted to beyond 2.15 A resolution at 100 K.

  19. Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis

    OpenAIRE

    Westfall, Corey S.; Sherp, Ashley M.; Zubieta, Chloe; Alvarez, Sophie; Schraft, Evelyn; Marcellin, Romain; Ramirez, Loren; Jez, Joseph M.

    2016-01-01

    In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenyl...

  20. Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings.

    Science.gov (United States)

    Lacey, Vanessa K; Louie, Gordon V; Noel, Joseph P; Wang, Lei

    2013-11-04

    Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. The nucleotide sequence of aroG, the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (phe) in Escherichia coli K 12

    OpenAIRE

    Davies, W.David; Davidson, Barrie E.

    1982-01-01

    We have determined the nucleotide sequence of aroG, the gene coding for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe), one of three isoenzymes that catalyse the first step of the biosynthesis of aromatic amino acids and vitamins in Escherichia coli K12. The DNA sequence agrees with previously published data on the N-terminal sequence, amino acid composition, and subunit molecular weight of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). There is significant identity i...

  2. An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

    Science.gov (United States)

    Achilli, Francesca; Bros-Facer, Virginie; Williams, Hazel P.; Banks, Gareth T.; AlQatari, Mona; Chia, Ruth; Tucci, Valter; Groves, Michael; Nickols, Carole D.; Seburn, Kevin L.; Kendall, Rachel; Cader, Muhammed Z.; Talbot, Kevin; van Minnen, Jan; Burgess, Robert W.; Brandner, Sebastian; Martin, Joanne E.; Koltzenburg, Martin; Greensmith, Linda; Nolan, Patrick M.; Fisher, Elizabeth M. C.

    2009-01-01

    SUMMARY Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, GarsC201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The GarsC201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons. PMID:19470612

  3. Upregulation of cystathionine-β-synthetase expression contributes to inflammatory pain in rat temporomandibular joint.

    Science.gov (United States)

    Miao, Xiuhua; Meng, Xiaowen; Wu, Geping; Ju, Zhong; Zhang, Hong-Hong; Hu, Shufen; Xu, Guang-Yin

    2014-02-03

    Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study was designed to investigate whether endogenous H2S synthesizing enzyme cystathionine-β-synthetase (CBS) plays a role in inflammatory pain in temporomandibular joint (TMJ). TMJ inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into TMJ of adult male rats. Von Frey filaments were used to examine pain behavioral responses in rats following injection of CFA or normal saline (NS). Whole cell patch clamp recordings were employed on acutely isolated TG neurons from rats 2 days after CFA injection. Western blot analysis was carried out to measure protein expression in TGs. Injection of CFA into TMJ produced a time dependent hyperalgesia as evidenced by reduced escape threshold in rats responding to VFF stimulation. The reduced escape threshold was partially reversed by injection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, in a dose-dependent manner. CFA injection led to a marked upregulation of CBS expression when compared with age-matched controls. CFA injection enhanced neuronal excitability as evidenced by depolarization of resting membrane potentials, reduction in rheobase, and an increase in number of action potentials evoked by 2 and 3 times rheobase current stimulation and by a ramp current stimulation of TG neurons innervating the TMJ area. CFA injection also led to a reduction of IK but not IA current density of TG neurons. Application of AOAA in TMJ area reduced the production of H2S in TGs and reversed the enhanced neural hyperexcitability and increased the IK currents of TG neurons. These data together with our previous report indicate that endogenous H2S generating enzyme CBS plays an

  4. Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis

    Directory of Open Access Journals (Sweden)

    van Helden Paul

    2010-05-01

    Full Text Available Abstract Background The assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH, in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria. Results It was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions. Conclusions The physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is it's activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity

  5. A WHEP Domain Regulates the Dynamic Structure and Activity of Caenorhabditis elegans Glycyl-tRNA Synthetase.

    Science.gov (United States)

    Chang, Chih-Yao; Chien, Chin-I; Chang, Chia-Pei; Lin, Bo-Chun; Wang, Chien-Chia

    2016-08-05

    WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases and play roles in tRNA or protein binding. We present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. The cytoplasmic form possessed an N-terminal WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence consisting of an mitochondrial targeting signal and an appended domain. Cross-species complementation assays showed that CeGRS1 effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 knock-out strain. Although both forms of CeGlyRS efficiently charged the cytoplasmic tRNAs(Gly) of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNA(Gly) isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA binding activity, deletion of this domain reduced the catalytic efficiency of the enzyme. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests a role for the WHEP domain as a regulator of the dynamic structure and activity of the enzyme. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Preparative synthesis of beta-L-malyl-coenzyme A assisted by malyl-coenzyme A synthetase from Pseudomonas AM1.

    Science.gov (United States)

    Willibald, B; Boves, H; Holler, E

    1995-05-20

    beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days. The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63% homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences. Yields of beta-L-[14C]malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column. For most biochemical work, the product was partially purified with an overall 45% yield by chromatography on DEAE-Sephacel. The identity of the compound as beta-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart. The enzymatic synthesis, especially of radioactively labeled beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis.

  7. Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Joyce, Michael A. [Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7 (Canada); Brownie, Edward R.; Hayakawa, Koto; Fraser, Marie E., E-mail: frasm@ucalgary.ca [Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4 (Canada); Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7 (Canada)

    2007-05-01

    Attempts to crystallize succinyl-CoA synthetase from the thermophile T. aquaticus were thwarted by proteolysis of the β-subunit and preferential crystallization of a truncated form. Crystals of the full-length enzyme were grown after the purification protocol was modified to include frequent additions of protease inhibitors. Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the β-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 Å resolution, are of the protein in complex with Mn{sup 2+}-GDP.

  8. Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy.

    Science.gov (United States)

    Jordanova, Albena; Irobi, Joy; Thomas, Florian P; Van Dijck, Patrick; Meerschaert, Kris; Dewil, Maarten; Dierick, Ines; Jacobs, An; De Vriendt, Els; Guergueltcheva, Velina; Rao, Chitharanjan V; Tournev, Ivailo; Gondim, Francisco A A; D'Hooghe, Marc; Van Gerwen, Veerle; Callaerts, Patrick; Van Den Bosch, Ludo; Timmermans, Jean-Pièrre; Robberecht, Wim; Gettemans, Jan; Thevelein, Johan M; De Jonghe, Peter; Kremensky, Ivo; Timmerman, Vincent

    2006-02-01

    Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

  9. Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

    Energy Technology Data Exchange (ETDEWEB)

    Iwaki, Jun [Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Suzuki, Ryuichiro [Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Research Centre for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566 (Japan); Fujimoto, Zui, E-mail: zui@affrc.go.jp; Momma, Mitsuru [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Kuno, Atsushi [Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560 (Japan); Research Centre for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566 (Japan); Hasegawa, Tsunemi [Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560 (Japan)

    2005-11-01

    Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K. Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.

  10. Molecular recognition of tryptophan tRNA by tryptophanyl-tRNA synthetase from Aeropyrum pernix K1.

    Science.gov (United States)

    Tsuchiya, Wataru; Hasegawa, Tsunemi

    2009-05-01

    The identity elements of transfer RNA are the molecular basis for recognition by each cognate aminoacyl-tRNA synthetase. In the archaea system, the tryptophan tRNA identity has not been determined in detail. To investigate the molecular recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase (TrpRS) from the hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, various mutant transcripts of tryptophan tRNA prepared by an in vitro transcription system were examined by overexpression of A. pernix TrpRS. Substitution of the discriminator base, A73, impaired tryptophan incorporation activity. Changing the G1-C72 base pair to other base pairs also decreased the aminoacylation activity. Substitutions of anticodon CCA revealed that the C34 and C35 mutants dramatically reduced aminoacylation with tryptophan, but the A36 mutants had the same activity as the wild type. The results indicate that the anticodon nucleotides C34, C35, discriminator base A73 and G1-C72 base pair are major recognition sites for A. pernix TrpRS.

  11. Genetic Code Optimization for Cotranslational Protein Folding: Codon Directional Asymmetry Correlates with Antiparallel Betasheets, tRNA Synthetase Classes

    Directory of Open Access Journals (Sweden)

    Hervé Seligmann

    2017-01-01

    Full Text Available A new codon property, codon directional asymmetry in nucleotide content (CDA, reveals a biologically meaningful genetic code dimension: palindromic codons (first and last nucleotides identical, codon structure XZX are symmetric (CDA = 0, codons with structures ZXX/XXZ are 5′/3′ asymmetric (CDA = −1/1; CDA = −0.5/0.5 if Z and X are both purines or both pyrimidines, assigning negative/positive (−/+ signs is an arbitrary convention. Negative/positive CDAs associate with (a Fujimoto's tetrahedral codon stereo-table; (b tRNA synthetase class I/II (aminoacylate the 2′/3′ hydroxyl group of the tRNA's last ribose, respectively; and (c high/low antiparallel (not parallel betasheet conformation parameters. Preliminary results suggest CDA-whole organism associations (body temperature, developmental stability, lifespan. Presumably, CDA impacts spatial kinetics of codon-anticodon interactions, affecting cotranslational protein folding. Some synonymous codons have opposite CDA sign (alanine, leucine, serine, and valine, putatively explaining how synonymous mutations sometimes affect protein function. Correlations between CDA and tRNA synthetase classes are weaker than between CDA and antiparallel betasheet conformation parameters. This effect is stronger for mitochondrial genetic codes, and potentially drives mitochondrial codon-amino acid reassignments. CDA reveals information ruling nucleotide-protein relations embedded in reversed (not reverse-complement sequences (5′-ZXX-3′/5′-XXZ-3′.

  12. Small-molecule inhibitors of the tuberculosis target, phenylalanyl-tRNA synthetase from Penicillium griseofulvum CPCC-400528

    Directory of Open Access Journals (Sweden)

    Li-Ning Wang

    2016-12-01

    Full Text Available Phenylalanyl-tRNA synthetase (PheRS, a member of aminoacyl-tRNA synthetase family, was the new target of anti-tubercular drug discovery. In an attempt to fully exploit the new potential anti-tuberculosis drugs presented in micro-organisms, we developed a high-throughput screening assay against Mycobacterium tuberculosis (Mtb PheRS and then screened a library consisting of 32,000 strains and 1500 natural product-derived compounds. One potent hit extract of Penicillium griseofulvum CPCC-400528 was identified. In this study, isopatulin (1, (+-epiepoformin (2 and gentisyl alcohol (3, three patulin-producing intermediates, together with three indole-tetramic acids, α-cyclopiazonic acid (4, β-cyclopiazonic acid (5 and iso-α-cyclopiazonic acid (6, were isolated and identified as bioactive constituents from P. griseofulvum CPCC-400528. Their structures were elucidated on the basis of spectroscopic data. Compounds 1, 3, 4, and 5 exhibited Mtb PheRS-inhibiting activities, as well as moderate to weak anti-tuberculosis activities against Mtb H37Rv.

  13. Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis.

    Science.gov (United States)

    Westfall, Corey S; Sherp, Ashley M; Zubieta, Chloe; Alvarez, Sophie; Schraft, Evelyn; Marcellin, Romain; Ramirez, Loren; Jez, Joseph M

    2016-11-29

    In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A thaliana This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants.

  14. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P.

    Science.gov (United States)

    Yanagisawa, Tatsuo; Sumida, Tomomi; Ishii, Ryohei; Takemoto, Chie; Yokoyama, Shigeyuki

    2010-09-01

    Aminoacyl-tRNA synthetase (aaRS) paralogs with unknown functions exist in various species. We now report novel 'protein lysylation' by an Escherichia coli lysyl-tRNA synthetase paralog, GenX/PoxA/YjeA. X-ray crystallographic analysis shows that the structure of the GenX protein resembles that of a class II aaRS. Further in vitro studies reveal that it specifically aminoacylates EF-P with lysine. The shape of the protein substrate mimics that of the L-shaped tRNA, and its lysylation site corresponds to the tRNA 3' end. Thus, we show how the aaRS architecture can be adapted to achieve aminoacylation of a specific protein. Moreover, in vivo analyses reveal that the translation elongation factor P (EF-P) lysylation by GenX is enhanced by YjeK (lysine 2,3-aminomutase paralog), which is encoded next to the EF-P gene, and might convert alpha-lysyl-EF-P to beta-lysyl-EF-P. In vivo analyses indicate that the EF-P modification by GenX and YjeK is essential for cell survival.

  15. Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study.

    Science.gov (United States)

    Nayak, Seema U; Griffiss, J McLeod; Blumer, Jeffrey; O'Riordan, Mary Ann; Gray, Wesley; McKenzie, Robin; Jurao, Robert A; An, Amanda T; Le, Melissa; Bell, Stacie J; Ochsner, Urs A; Jarvis, Thale C; Janjic, Nebojsa; Zenilman, Jonathan M

    2017-08-01

    Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.). Copyright © 2017 American Society for Microbiology.

  16. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  17. In vivo footprinting of the carbamoylphosphate synthetase I cAMP-response unit indicates important roles for FoxA and PKA in formation of the enhanceosome

    NARCIS (Netherlands)

    Hoogenkamp, M.; Stallen, J. M. P.; Lamers, W. H.; Gaemers, I. C.

    2006-01-01

    The expression of carbamoylphosphate synthetase-I (CPS), the first and rate-determining enzyme of the urea cycle, is regulated at the transcriptional level by glucocorticoids and glucagon, the latter acting via cyclic AMP (cAMP). The hormonal response is mediated by a distal enhancer located 6.3 kb

  18. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.

    1994-01-01

    Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically...

  19. scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

    NARCIS (Netherlands)

    Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M

    1996-01-01

    A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high

  20. In vivo study of developmental changes in carbamoyl-phosphate synthetase I in rat liver. Repression of the enzyme synthesis immediately after birth.

    Science.gov (United States)

    Murakami, A; Kitagawa, Y; Sugimoto, E

    1983-01-20

    The regulatory mechanism of the developmental increase of carbamoyl-phosphate synthetase I in fetal and neonatal rat liver was studied in vivo. The appearance and rapid increase of the enzyme in late fetal period were caused by de novo synthesis of the enzyme protein. The amount of the enzyme protein analyzed by SDS-polyacrylamide gel electrophoresis was proportional to the enzyme activity throughout the period of development. No indication was observed for preexisting protein which could be converted into the active protein. A novel system for the in vivo study of carbamoyl-phosphate synthetase I synthesis was developed. Hepatocytes, mechanically dispersed by repeated passage of the tissue through a pipet, incorporated [35S]methionine into the enzyme. Taking advantage of this system, the regulation of the enzyme synthesis was studied. In vivo synthesis of the enzyme was detected at 4 days before birth and rapidly increased until 1 day before birth. However, the enzyme synthesis was markedly repressed after birth, when the amount of carmamoyl-phosphate synthetase I itself reached the adult level. This result was in a clear contrast with the constant level of the translatable mRNA (Raymond, Y. and Shore, G.C. (1981) Biochim. Biophys. Acta 656, 111-119) and suggested that post-transcriptional regulation is important in addition to the level of mRNA for the regulation of the carbamoyl-phosphate synthetase I level.

  1. Cytosolic glutamine synthetase Gln1;2 is the main isozyme contributing to GS1 activity and can be up-regulated to relieve ammonium toxicity

    DEFF Research Database (Denmark)

    Guan, Miao; de Bang, Thomas Christian; 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 th...

  2. Genetic Diversity of Polyketide Synthase/Nonribosomal Peptide Synthetase Genes in Isolates of the Barley Net Blotch Fungus Pyrenophora teres f. teres

    Science.gov (United States)

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes responsible for biosynthesis of diverse small molecules (e.g., mycotoxins and phytotoxins) in filamentous ascomycetes. Both PKS and NRPS genes are present in fungal genomes as large gene families but...

  3. Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

    Directory of Open Access Journals (Sweden)

    Iyer Lakshminarayan M

    2010-08-01

    Full Text Available Abstract Background Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS in non-ribosomal peptide ligation. Results Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS. We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains. Conclusions The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the

  4. Inhibition by propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia.

    Science.gov (United States)

    Coude, F X; Sweetman, L; Nyhan, W L

    1979-01-01

    In the search for the mechanism by which hyperammonemia complicates propionic and methylmalonic acidemia the effects of a series of acyl-coenzyme A (CoA) derivatives were studied on the activity of N-acetylglutamate synthetase in rat liver mitochondria using acetyl-CoA as substrate. Propionyl-CoA was found to be a competitive inhibitor. The inhibition constant of 0.71 mM is in the range of concentrations of propionate found in the serum of patients with propionic and methylmalonic acidemia. Propionyl-CoA was also found to be a substrate for N-acetylglutamate synthetase, forming N-propionylglutamate. This compound was a weak activator of rat liver carbamoylphosphate synthetase; the activation constant was 1.1 mM as compared with 0.12 mM for N-acetylglutamate. A decreased level of N-acetylglutamate in liver mitochondria that would follow inhibition of N-acetylglutamate synthetase by propionyl-CoA would be expected to lead to hyperammonemia. Methylmalonyl-CoA, tiglyl-CoA, and isovaleryl-CoA at a concentration of 3 mM caused 30-70% inhibition of N-acetylglutamate synthetase. 3the latter two compounds are readily detoxified by the formation of N-acylglycine conjugates in liver, which may prevent large accumulations and could explain why hyperammonemia is not characteristic of patients with beta-ketothiolase deficiency or isovaleric acidemia in whom these compounds would be expected to be elevated. PMID:500823

  5. The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular Utilization

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Black, P N; Zhao, X D

    2001-01-01

    Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p...... enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2). These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization...

  6. The implication of dihydrofolate reductase and dihydropteroate synthetase gene mutations in modification of Plasmodium falciparum characteristics

    DEFF Research Database (Denmark)

    A-Elbasit, Ishraga E; Alifrangis, Michael; Khalil, Insaf F

    2007-01-01

    BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are enzymes of central importance in parasite metabolism. The dhfr and dhps gene mutations are known to be associated with sulphadoxine/pyrimethamine (SP) resistance. OBJECTIVE: To investigate...... the effects of dhfr/dhps mutations on parasite characteristics other than SP resistance. METHOD: Parasite infections obtained from 153 Sudanese patients with uncomplicated falciparum malaria treated with SP or SP + chloroquine, were successfully genotyped at nine codons in the dhfr/dhps genes by PCR......-ELISA. RESULTS & CONCLUSION: Mutations were detected in dhfr at N51I, S108N and C59R, and in at dhps at A/S436F, A437G, K540E and A581G, the maximum number of mutations per infection were five. Based on number of mutant codons per infection (multiplicity of mutation, MOM), the infections were organized into six...

  7. The Cell Wall Teichuronic Acid Synthetase (TUAS Is an Enzyme Complex Located in the Cytoplasmic Membrane of Micrococcus luteus

    Directory of Open Access Journals (Sweden)

    Lingyi Lynn Deng

    2010-01-01

    composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6α-D-Glcp−1-]n, which is covalently anchored to the peptidoglycan on the inner cell wall and extended to the outer surface of the cell envelope. An enzyme complex responsible for the TUA chain biosynthesis was purified and characterized. The 440 kDa enzyme complex, named teichuronic acid synthetase (TUAS, is an octomer composed of two kinds of glycosyltransferases, Glucosyltransferase, and ManNAcA-transferase, which is capable of catalyzing the transfer of disaccharide glycosyl residues containing both glucose and the N-acetylmannosaminuronic acid residues. TUAS displays hydrophobic properties and is found primarily associated with the cytoplasmic membrane. The purified TUAS contains carotinoids and lipids. TUAS activity is diminished by phospholipase digestion. We propose that TUAS serves as a multitasking polysaccharide assembling station on the bacterial membrane.

  8. Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

    Science.gov (United States)

    Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

    2013-04-02

    Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Fluorescence study on ligand induced conformational changes of glutamine synthetase from Bacillus brevis Bb G1 under sporulating conditions

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    SUJA ABRAHAM

    2015-04-01

    Full Text Available Glutamine synthetase, an important enzyme of nitrogen metabolism, was purified under sporulating conditions (GSala. The effect of ligands on the tryptophan fluorescence of the purified enzyme GSala was investigated. With increasing concentrations of L-glutamine in GSala, a blue shift in emission maximum with an increase in fluorescence intensity and decrease in life times were observed compared to the emission maximum, fluorescence intensity and life times of GSala. With increasing concentrations of glycine in GSala, a shift in emission maximum, change in fluorescence intensity and change in lifetimes were observed compared to the emission maximum, fluorescence intensity and life times of GSala. These observations strongly support the possibility that GSala undergoes a conformational change on binding with ligands and each ligand produced different conformational changes in GSala. Also, different concentrations of each ligand produced different protein conformations in the enzyme GSala.

  10. Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

    DEFF Research Database (Denmark)

    Paul, David S; Grevengoed, Trisha J; Pascual, Florencia

    2014-01-01

    In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1(H-/-)), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy...... of sarco/endoplasmic reticulum calcium ATPase and phospholamban showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1(H-/-) mice with rapamycin. Six to eight week old Acsl1(H-/-) mice and their littermate controls were given i.......p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin...

  11. Crystallization and preliminary crystallographic studies of putative threonyl-tRNA synthetases from Aeropyrum pernix and Sulfolobus tokodaii.

    Science.gov (United States)

    Shimizu, Satoru; Juan, Ella Czarina Magat; Miyashita, Yu Ichiro; Sato, Yoshiteru; Hoque, Md Mominul; Suzuki, Kaoru; Yogiashi, Masataka; Tsunoda, Masaru; Dock-Bregeon, Anne Catherine; Moras, Dino; Sekiguchi, Takeshi; Takénaka, Akio

    2008-10-01

    Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing the aminoacylation of tRNA(Thr) and editing misacylation. ThrRS generally contains an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. The sequences of the editing domain in ThrRSs from archaea differ from those in bacteria and eukaryotes. Furthermore, several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7 contain two genes encoding either the catalytic or the editing domain of ThrRS. To reveal the structural basis for this evolutionary divergence, the two types of ThrRS from the crenarchaea A. pernix and S. tokodaii have been overexpressed in Eschericha coli, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected and the structure of a selenomethionine-labelled A. pernix type-1 ThrRS crystal has been solved using the MAD method.

  12. Associations of antifolate resistance in vitro and point mutations in dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum

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    Biswas S

    2004-01-01

    Full Text Available Background: Antifolate antimalarials like sulfadoxine-pyrimethamine are used as second-line treatment for Plasmodium falciparum malaria patients who fail to respond to chloroquine. The efficacy of the sulfa-pyrimethamine combination in the treatment is also compromised by the development of resistance in the parasite. Resistance to these drugs has been shown to encode with point mutations in dihydrofolate reductase and dihydropteroate synthetase genes. Settings: An experimental study. Maerial and methods: Forty clinical isolates collected from different geographical locations in India were used to assess the relationships between resistance to sulfadoxine-pyrimethamine (SP and mutations in P. falciparum dihydrofolate reductase (DHFR and dihydropteroate synthetase (DHPS. In vitro drug susceptibility and mutation-specific polymerase chain reaction (PCR assays were also done. Results: It was observed that a number of isolates possessed mutant genotypes and showed low sensitivity to SP in vitro. Of the 40 clinical isolates studied, 87.5% had DHFR and 15% had DHPS gene mutations. As observed from PCR results, 55( (22/40 presented double mutation of DHFR Arg-59 and Asn-108 and 32.5 % (13/40 had single mutant type allele of Asn-108. Of the 40 isolates, 10 % (4/40 presented doubly mutated forms of DHPS Phe-436 and Thr-613 and single mutant type allele Gly-581 was detected in 5 % (2/40 isolates. Parasites carrying double or single mutant forms of DHFR/DHPS showed elevated minimum inhibitory concentration (MIC values of both pyrimethamine (760-6754 nM; r=0.69 and sulfadoxine (108 - 540 mM; r=0.87 when compared to sensitive and resistant strains. Conclusion: Though there was a correlation between molecular techniques and in vitro drug sensitivity profiles, the relevance of these findings to the clinical efficacy of SP combination drugs needs to be established by controlled clinical trials.

  13. The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma

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    Angkana Thongkum

    2017-06-01

    Full Text Available Argininosuccinate synthetase (ASS, a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC. Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(- tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20, which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU in ASS(-HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(- while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+ cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP, myeloid leukemia cell differentiation protein (Mcl-1 and B-cell lymphoma-2 (Bcl-2. Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD and thymidylate synthase (TS and malate dehydrogenase-1 (MDH-1 in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-hepatoma and warrants further clinical investigation.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.

    Science.gov (United States)

    Horsfall, Louise E; Nelson, Adam; Berry, Alan

    2010-07-01

    Sialylated oligosaccharides, present on mammalian outer-cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host's immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages, the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP-Neu5Ac synthetase (CNS). Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine, and the steady-state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction, pyrophosphate. Mutations that caused the greatest loss in activity included K142A, D211A, D209A and a series of mutations at residue Q104, highlighted from sequence-alignment studies of related enzymes, demonstrating significant roles for these residues in the catalytic mechanism of CNS. The mutations of D211A and D209A provide strong evidence for a previously proposed metal-binding site in the enzyme, and the results of our mutations at residue Q104 lead us to include this residue in the metal-binding site of an intermediate complex. This suggests that, like the sugar-activating lipopolysaccharide-synthesizing CMP-2-keto-3-deoxy-manno-octonic acid synthetase enzyme KdsB, CNS recruits two Mg(2+) ions during the catalytic cycle.

  16. Common and distinct clinical features in adult patients with anti-aminoacyl-tRNA synthetase antibodies: heterogeneity within the syndrome.

    Directory of Open Access Journals (Sweden)

    Yasuhito Hamaguchi

    Full Text Available OBJECTIVE: To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs. METHODS: This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009. RESULTS: Anti-ARS Ab specificity included anti-Jo-1 (36%, anti-EJ (23%, anti-PL-7 (18%, anti-PL-12 (11%, anti-KS (8%, and anti-OJ (5%. These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM-specific skin manifestations (heliotrope rash and Gottron's sign were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset. CONCLUSION: Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the "anti-synthetase syndrome."

  17. Fatty acid transport protein 4 is the principal very long chain fatty acyl-CoA synthetase in skin fibroblasts.

    Science.gov (United States)

    Jia, Zhenzhen; Moulson, Casey L; Pei, Zhengtong; Miner, Jeffrey H; Watkins, Paul A

    2007-07-13

    Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54-64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.

  18. Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase.

    Science.gov (United States)

    Chen, Qingfeng; Westfall, Corey S; Hicks, Leslie M; Wang, Shiping; Jez, Joseph M

    2010-09-24

    The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg(2+) or Mn(2+) for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4-9-fold reductions in k(cat)/K(m) relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.

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

    Science.gov (United States)

    Hawkins, Aaron S.; Han, Yejun; Bennett, Robert K.; Adams, Michael W. W.; Kelly, Robert M.

    2013-01-01

    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 H2-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 (Km values of 1.9 and 1.5 mm for Msed_0406 and Msed_0394, respectively) but with different rates (1.69 and 0.22 μmol × min−1 × 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. PMID:23258541

  20. Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases.

    Science.gov (United States)

    Yu, Hai; Yu, Hui; Karpel, Rebekah; Chen, Xi

    2004-12-15

    Three C terminal His6-tagged recombinant microbial CMP-sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP-sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP-sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP-sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100-200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.

  1. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

    1989-01-01

    by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence......The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed......: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6...

  2. Striatal N-Acetylaspartate Synthetase Shati/Nat8l Regulates Depression-Like Behaviors via mGluR3-Mediated Serotonergic Suppression in Mice

    Science.gov (United States)

    Miyamoto, Yoshiaki; Iegaki, Noriyuki; Fu, Kequan; Ishikawa, Yudai; Sumi, Kazuyuki; Azuma, Sota; Uno, Kyosuke; Muramatsu, Shin-ichi

    2017-01-01

    Abstract Background Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes N-acetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3

  3. Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species.

    Science.gov (United States)

    Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Takahashi-Nakaguchi, Azusa; Matsuzawa, Tetsuhiro; Suzuki, Ken-ichiro; Fujita, Nobuyuki; Gonoi, Tohru

    2014-04-30

    Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology. Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text. We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied

  4. Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain.

    OpenAIRE

    Oliver, C N; Starke-Reed, P E; Stadtman, E. R.; Liu,G.J.; Carney, J M; Floyd, R A

    1990-01-01

    Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed...

  5. A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.

    Directory of Open Access Journals (Sweden)

    Christine Böttcher

    Full Text Available 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-ylethyl]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 K(i-values 17-68-fold lower than the respective K(m-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.

  6. Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes

    Science.gov (United States)

    Lorenzi, Adriana Sturion; Silva, Genivaldo Gueiros Z.; Lopes, Fabyano Alvares Cardoso; Chia, Mathias Ahii; Edwards, Robert A.

    2016-01-01

    Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes). PMID:27151783

  7. Reinvestigation of aminoacyl-tRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex.

    Directory of Open Access Journals (Sweden)

    Kyutae Kim

    Full Text Available Twenty different aminoacyl-tRNA synthetases (ARSs link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS, together with three ARS-interacting multifunctional proteins (AIMPs, are currently known to assemble the multi-synthetase complex (MSC. However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2 was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.

  8. Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

    Directory of Open Access Journals (Sweden)

    Antonella Albino

    2011-09-01

    Full Text Available Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from L-γ-glutamyl-L-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P212121, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.

  9. Modulating plant primary amino acid metabolism as a necrotrophic virulence strategy: the immune-regulatory role of asparagine synthetase in Botrytis cinerea-tomato interaction.

    Science.gov (United States)

    Seifi, Hamed; De Vleesschauwer, David; Aziz, Aziz; Höfte, Monica

    2014-01-01

    The fungal plant pathogen Botrytis cinerea is the causal agent of the "gray mold" disease on a broad range of hosts. As an archetypal necrotroph, B. cinerea has evolved multiple virulence strategies for inducing cell death in its host. Moreover, progress of B. cinerea colonization is commonly associated with induction of senescence in the host tissue, even in non-invaded regions. In a recent study, we showed that abscisic acid deficiency in the sitiens tomato mutant culminates in an anti-senescence defense mechanism which effectively contributes to resistance against B. cinerea infection. Conversely, in susceptible wild-type tomato a strong induction of senescence could be observed following B. cinerea infection. Building upon this earlier work, we here discuss the immune-regulatory role of a key senescence-associated protein, asparagine synthetase. We found that infection of wild-type tomato leads to a strong transcriptional upregulation of asparagine synthetase, followed by a severe depletion of asparagine titers. In contrast, resistant sitiens plants displayed a strong induction of asparagine throughout the course of infection. We hypothesize that rapid activation of asparagine synthetase in susceptible tomato may play a dual role in promoting Botrytis cinerea pathogenesis by providing a rich source of N for the pathogen, on the one hand, and facilitating pathogen-induced host senescence, on the other.

  10. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix.

    Science.gov (United States)

    Ahmad, Sadeem; Sravankumar, Antony S K; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2012-11-01

    The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P4(1)2(1)2 or P4(3)2(1)2 space group and consist of one monomer per asymmetric unit.

  11. E. coli glutamyl-tRNA synthetase is inhibited by anticodon stem-loop domains and a minihelix.

    Science.gov (United States)

    Gustilo, Estella M; Dubois, Daniel Y; Lapointe, Jacques; Agris, Paul F

    2007-07-01

    Portions of E. coli tRNA(Glu) having identity determinants for glutamyl-tRNA synthetase (ERS, EC 6.1.1.17) have been designed to be the first RNA inhibitors of a Class I synthetase. ERS recognizes the 2-thionyl group of 2-thio-5-methylaminomethyluridine (mnm(5)s(2)U(34)) in the first or wobble anticodon position of E. coli tRNA(Glu). The interaction, as revealed by structural analysis, though specific, appears tenuous. Thus, it is surprising that RNAs designed from this tRNA's anticodon stem and loop domain with (ASL(Glu)-s(2)U(34)) and without s(2)U(34) are bound by ERS and inhibit aminoacylation of the native tRNA. ASL(Glu), ASL(Glu)-s(2)U(34), and a minihelix(Glu) composed of identity determinants of the amino acid accepting stem were thermally stable under conditions of aminoacylation (T(m)s = 75 +/- 1.5, 76 +/- 0.9 and 83 +/- 2.0 degrees C, respectively). In binding competition, the modified ASL(Glu)-s(2)U(34) bound ERS with a higher affinity (half maximal inhibiting concentration, IC(50), 5.1 +/- 0.4 microM) than its unmodified counterpart, ASL(Glu) (IC(50), 10.3 +/- 0.6 microM). The minihelix(Glu), ASL(Glu)-s(2)U(34) and ASL(Glu) bound ERS with K(d)s of 9.9 +/- 3.3, 6.5 +/- 1.7 and 20.5 +/- 3.8 microM. ERS aminoacylation of tRNA(Glu) was inhibited by the tRNA fragments. Unmodified ASL(Glu), minihelix(Glu), and ASL(Glu)-s(2)U(34) exhibited a K(ic) of 1.9 +/- 0.2 microM, 4.1 +/- 0.2 microM, and 6.5 +/- 0.1 microM, respectively. The modified ASL(Glu)-s(2)U(34), though having a higher affinity for ERS, may be released more readily and thus, not be as good an inhibitor as the unmodified ASL. Thus, the RNA constructs are effective tools to study RNA-protein interaction.

  12. Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum.

    Science.gov (United States)

    Peterson, P J; Fowden, L

    1965-10-01

    1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus

  13. Entamoeba histolytica acetyl–CoA synthetase: biomarker of acute amoebic liver abscess

    Directory of Open Access Journals (Sweden)

    Lim Boon Huat

    2014-06-01

    Conclusions: This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

  14. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    Science.gov (United States)

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi.

  15. The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

    Energy Technology Data Exchange (ETDEWEB)

    Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf; Neuhof, Torsten; Baker, Scott E.; Druzhinina, Irina S.; Kubicek, Christian P.; Brückner, Hans; von Dohren, Hans

    2012-03-01

    The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.

  16. Proteomic interrogation of androgen action in prostate cancer cells reveals roles of aminoacyl tRNA synthetases.

    Directory of Open Access Journals (Sweden)

    Adaikkalam Vellaichamy

    2009-09-01

    Full Text Available Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer.

  17. A Conserved Proline Triplet in Val-tRNA Synthetase and the Origin of Elongation Factor P

    Directory of Open Access Journals (Sweden)

    Agata L. Starosta

    2014-10-01

    Full Text Available Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNAVal with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNAVal with valine and preventing formation of mischarged Thr-tRNAVal as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.

  18. Coupling native page/activity-staining with SDS-PAGE/immunodetection for the analysis of glutamine synthetase isoforms in spinach

    Directory of Open Access Journals (Sweden)

    Dragićević M.

    2011-01-01

    Full Text Available Glutamine synthetase (GS is a key nitrogen-assimilating enzyme in plants and a target for the broad-spectrum herbicide glufosinate. Understanding its kinetic and structural properties is of major agricultural importance. Spinach (Spinacia oleracea is classified as a plant expressing only chloroplastic GS activity. We have analyzed soluble proteins in the spinach by coupling native polyacrylamide gel electrophoresis (PAGE-activity detection, based on phosphate precipitation, with SDS-PAGE/immunoblotting. One cytosolic (GS1 isoform from the roots and two chloroplastic (GS2 isoforms expressed in leaves were resolved by native PAGE. The identity of the obtained bands was established by the application of GS-specific inhibitors, L-methionine sulfoximine and glufosinate. Examination by sodium dodecyl sulfate (SDS-PAGE/ Western analysis with anti-GS antibodies, confirmed the identity of the active bands and revealed that both chloroplastic isoforms are composed of 44 kDa subunits, while the cytosolic isoform consists of 40 kDa subunits. The presence of more GS2 isozymes than encoded in the spinach genome is discussed in terms of posttranslational modifications.

  19. New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

    Energy Technology Data Exchange (ETDEWEB)

    Gallo, A.; Bruno, K. S.; Solfrizzo, M.; Perrone, G.; Mule, G.; Visconti, A.; Baker, S. E.

    2012-09-14

    Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.

  20. CDP-diacylglycerol synthetase coordinates cell growth and fat storage through phosphatidylinositol metabolism and the insulin pathway.

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2014-03-01

    Full Text Available During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA, which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.

  1. CDP-diacylglycerol synthetase coordinates cell growth and fat storage through phosphatidylinositol metabolism and the insulin pathway.

    Science.gov (United States)

    Liu, Yuan; Wang, Wei; Shui, Guanghou; Huang, Xun

    2014-03-01

    During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.

  2. Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.

    Science.gov (United States)

    Sungwienwong, Itthipol; Hostetler, Zachary M; Blizzard, Robert J; Porter, Joseph J; Driggers, Camden M; Mbengi, Lea Z; Villegas, José A; Speight, Lee C; Saven, Jeffery G; Perona, John J; Kohli, Rahul M; Mehl, Ryan A; Petersson, E James

    2017-05-03

    The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.

  3. Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

    Science.gov (United States)

    Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

    2015-08-01

    Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

    Science.gov (United States)

    Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

    2016-08-02

    Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

  5. Biochemical and structural characterization of the GTP-preferring succinyl-CoA synthetase from Thermus aquaticus.

    Science.gov (United States)

    Joyce, Michael A; Hayakawa, Koto; Wolodko, William T; Fraser, Marie E

    2012-07-01

    Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two β-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn(2+). A water molecule and Pro20β in T. aquaticus take the place of Gln20β in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.

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

    Science.gov (United States)

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

    2011-11-01

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

  7. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases.

    Science.gov (United States)

    Miller, Bradley R; Sundlov, Jesse A; Drake, Eric J; Makin, Thomas A; Gulick, Andrew M

    2014-10-01

    Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. © 2014 Wiley Periodicals, Inc.

  8. Analysis of the Linker Region Joining the Adenylation and Carrier Protein Domains of the Modular Non-Ribosomal Peptide Synthetases

    Science.gov (United States)

    Miller, Bradley R.; Sundlov, Jesse A.; Drake, Eric J.; Makin, Thomas A.; Gulick, Andrew M.

    2014-01-01

    Non-Ribosomal Peptide Synthetases (NRPSs) are multi-modular proteins capable of producing important peptide natural products. Using an assembly-line process the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal sub-domain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. PMID:24975514

  9. The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

    Science.gov (United States)

    Cheng, Tai-Sheng

    2012-11-15

    The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2 mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H(2)O(2) and NBT stain for superoxide radicals (O(2)(·-)), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M. (UMM); (HWMRI)

    2016-09-05

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

  11. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture.

    Science.gov (United States)

    Miller, Bradley R; Drake, Eric J; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

    2016-10-21

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Carbamoyl phosphate synthetase subunit MoCpa2 affects development and pathogenicity by modulating arginine biosynthesis in Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Xinyu Liu

    2016-12-01

    Full Text Available Arginine is a semi-essential amino acid that affects physiological and biochemical functions. The CPA2 gene in yeast encodes a large subunit of arginine-specific carbamoyl phosphate synthetase and is involved in arginine biosynthesis. Here, an ortholog of yeast CPA2 was identified in the rice blast fungus Magnaporthe oryzae, and was named MoCPA2. MoCpa2 is an 1180-amino acid protein which contains an ATP grasp domain and two CPSase domains. Targeted deletion of MoCPA2 supported its role in de novo arginine biosynthesis in M. oryzae as mutant phenotypes were complemented by arginine but not ornithine. The ΔMocpa2 mutant exhibited defects in asexual development and pathogenicity but not appressorium formation. Further examination revealed that the invasive hyphae of the ΔMocpa2 mutant were restricted mainly to the primary infected cells. In addition, the ΔMocpa2 mutant was unable to induce a plant defense response and had the ability to scavenge ROS during pathogen-plant interactions. Structure analysis revealed that the ATP grasp domain and each CPS domain were indispensable for the proper localization and full function of MoCpa2. In summary, our results indicate that MoCpa2 plays an important role in arginine biosynthesis, and affects growth, conidiogenesis, and pathogenicity. These results suggest that research into metabolism and processes that mediate amino acid synthesis are valuable for understanding M. oryzae pathogenesis.

  13. The Folylpolyglutamate Synthetase Plastidial Isoform Is Required for Postembryonic Root Development in Arabidopsis1[W][OA

    Science.gov (United States)

    Srivastava, Avinash C.; Ramos-Parra, Perla A.; Bedair, Mohamed; Robledo-Hernández, Ana L.; Tang, Yuhong; Sumner, Lloyd W.; Díaz de la Garza, Rocío I.; Blancaflor, Elison B.

    2011-01-01

    A recessive Arabidopsis (Arabidopsis thaliana) mutant with short primary roots and root hairs was identified from a forward genetic screen. The disrupted gene in the mutant encoded the plastidial isoform of folylpolyglutamate synthetase (FPGS), previously designated as AtDFB, an enzyme that catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The short primary root of atdfb was associated with a disorganized quiescent center, dissipated auxin gradient in the root cap, bundled actin cytoskeleton, and reduced cell division and expansion. The accumulation of monoglutamylated forms of some folate classes in atdfb was consistent with impaired FPGS function. The observed cellular defects in roots of atdfb underscore the essential role of folylpolyglutamates in the highly compartmentalized one-carbon transfer reactions (C1 metabolism) that lead to the biosynthesis of compounds required for metabolically active cells found in the growing root apex. Indeed, metabolic profiling uncovered a depletion of several amino acids and nucleotides in atdfb indicative of broad alterations in metabolism. Methionine and purines, which are synthesized de novo in plastids via C1 enzymatic reactions, were particularly depleted. The root growth and quiescent center defects of atdfb were rescued by exogenous application of 5-formyl-tetrahydrofolate, a stable folate that was readily converted to metabolically active folates. Collectively, our results indicate that AtDFB is the predominant FPGS isoform that generates polyglutamylated folate cofactors to support C1 metabolism required for meristem maintenance and cell expansion during postembryonic root development in Arabidopsis. PMID:21233333

  14. A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

    Science.gov (United States)

    Kojima, Kouji; Keta, Sumie; Uesaka, Kazuma; Kato, Akihiro; Takatani, Nobuyuki; Ihara, Kunio; Omata, Tatsuo; Aichi, Makiko

    2016-12-01

    Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.

  15. Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase.

    Science.gov (United States)

    Chopra, Shaileja; Palencia, Andrés; Virus, Cornelia; Tripathy, Ashutosh; Temple, Brenda R; Velazquez-Campoy, Adrian; Cusack, Stephen; Reader, John S

    2013-01-01

    Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA(Leu) for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3'-end. Our findings reveal an intriguing tRNA-dependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design.

  16. Bacterial resistance to leucyl-tRNA synthetase inhibitor GSK2251052 develops during treatment of complicated urinary tract infections.

    Science.gov (United States)

    O'Dwyer, Karen; Spivak, Aaron T; Ingraham, Karen; Min, Sharon; Holmes, David J; Jakielaszek, Charles; Rittenhouse, Stephen; Kwan, Alan L; Livi, George P; Sathe, Ganesh; Thomas, Elizabeth; Van Horn, Stephanie; Miller, Linda A; Twynholm, Monique; Tomayko, John; Dalessandro, Marybeth; Caltabiano, Madelyn; Scangarella-Oman, Nicole E; Brown, James R

    2015-01-01

    GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening.

    Science.gov (United States)

    Shapiro, Adam B; Plant, Helen; Walsh, Jarrod; Sylvester, Mark; Hu, Jun; Gao, Ning; Livchak, Stephania; Tentarelli, Sharon; Thresher, Jason

    2014-09-01

    A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series. © 2014 Society for Laboratory Automation and Screening.

  18. Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

    2014-03-01

    Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

  19. Streptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene.

    Science.gov (United States)

    de Crécy-Lagard, V; Saurin, W; Thibaut, D; Gil, P; Naudin, L; Crouzet, J; Blanc, V

    1997-01-01

    Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations. PMID:9303382

  20. Drosophila ortholog of succinyl-CoA synthetase {beta} subunit: a novel modulator of Drosophila KCNQ channels.

    Science.gov (United States)

    Gao, Lei; Fei, Hong; Connors, Nathan C; Zhang, Jiaming; Levitan, Irwin B

    2008-05-01

    Voltage-gated KCNQ potassium channels are responsible for slowly activating potassium currents in heart, brain, and other tissues. Functional defects of KCNQ channels are linked with many diseases, including epilepsy and cardiac arrhythmias. Therefore KCNQ potassium channels have been widely studied, especially in the CNS. We have identified Drosophila CG11963, which encodes a protein orthologous to the beta subunit of mammalian succinyl-CoA synthetase (SCS, also known as succinate thiokinase), as a novel modulator of Drosophila KCNQ channels. Direct interaction of CG11963 and dKCNQ was demonstrated by yeast two-hybrid screen and coimmunoprecipitation. Cell surface biotinylation experiments further confirmed that CG11963 resides on the plasma membrane of tsA-201 cells. Coexpression of CG11963 with dKCNQ shifts the conductance-voltage (G-V) relationship of dKCNQ channels to more positive membrane potentials in Chinese hamster ovary (CHO) cells. Moreover, directly dialyzing glutathione S-transferase fusion CG11963 protein into CHO cells also shifts the dKCNQ G-V curve rightward. The effect of CG11963 persists in the presence of 1 mM adenosine triphosphate (ATP), a substrate of SCS. Taken together, our data define CG11963 as a new dKCNQ-binding protein capable of modulating the properties of the channel. Our evidence suggests that this modulation is mediated by direct interaction of CG11963 with the channel and is not dependent on ATP.

  1. Glial fibrillary acidic protein and glutamine synthetase in subregions of prefrontal cortex in schizophrenia and mood disorder.

    Science.gov (United States)

    Toro, Carla T; Hallak, Jaime E C; Dunham, Jason S; Deakin, John F W

    2006-09-01

    Several theories of schizophrenia suggest dysfunction in glutamate neurotransmission in higher brain regions such as the prefrontal cortex (PFC). Previous studies have investigated whether astroglial abnormalities could give rise to glutamate dysfunction using glial fibrillary acidic protein (GFAP) immunocytochemistry. We have used quantitative immunoautoradiography to measure glutamine synthetase (GS), the glial enzyme which recycles synaptic glutamate, as a more direct test of glial mechanisms of abnormal glutamate function in schizophrenia. We compared GS with GFAP immunoautoradiography in dorsolateral (area 9) and orbitofrontal (area 11/47) cortex. Optical density measures from film autoradiographs revealed an increase in GFAP immunoreactivity in area 9 in schizophrenia and a decrease in area 11/47 in both schizophrenia and bipolar disorder. The increase in GFAP in area 9 significantly correlated with lifetime antipsychotic drug treatment, whereas the reduction in area 11/47 occurred despite this effect. There were no changes in GS immunoreactivity in any psychiatric disorder. Regional and antigen-specific down-regulation of GFAP protein in OFC in schizophrenia and bipolar disorder may relate to disease mechanisms of psychosis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Knight, T.J. [Univ. of Southern Maine, Portland, ME (United States); Temple, S.; Sengupta-Gopalan, C. [New Mexico State Univ., Las Curces, NM (United States)] [and others

    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.

  3. Аldosterone synthetase gene (CYP11B2 polymorphism and structural parameters of the left ventricle in patients with coronary heart disease, postinfarction cardiosclerosis

    Directory of Open Access Journals (Sweden)

    M. N. Dolzhenko

    2017-12-01

    Full Text Available Purpose of the work – to investigate the possible contribution of aldosterone synthetase gene (CYP11B2 polymorphism to the disease course and structural parameters of LV in patients with coronary heart disease, postinfarction cardiosclerosis. Materials and мethods. General clinical examination of 100 patients with postinfarction cardiosclerosis was done at the Cardiology Department of P. L Shupyk NMAPE. Genetic testing was performed by polymerase chain reaction in real time at the Bogomolets Institute of Physiology,Kyiv,Ukraine. Exclusion criteria were hemodynamically significant valvular heart diseases, chronic obstructive pulmonary diseases, permanent or temporary heart pacing, acute heart failure and implanted cardioverter-defibrillator, permanent atrial fibrillation. Statistical analysis of the results was performed using Microsoft Excel, the statistical program SPSS (version 20, US. The results obtained are presented as M ± σ. Results. The stenosis of the left main coronary artery was observed in 25.9 % of cases in the subgroup of the TT variant. It should be noted that in the TC subgroup of aldosterone synthase gene variant polymorphism the incidence of the left main coronary artery lesion was 13.9 %. There has been no single case of left main coronary artery lesion in the SS subgroup with little statistical significance in comparison with the subgroup of TT variant of the polymorphism (P = 0.048. In the analysis of clinical data the most marked manifestations of angina pectoris were in subgroups of TT and TC – 73.3 % and 72.7 %, respectively, compared with CC subgroup – 40 %, reliable for both subgroups (P1.2 = 0.95, P1.3 = 0.039, P2.3 = 0.029. In the analysis of LV morphological characteristics the smallest indices of the LV mass have been revealed in the CC subgroup of the polymorphism variant (190.5 ± 52.1 g, compared with the LV mass values in the TT subgroup (231.00 ± 55.21 g, P = 0.03 and TC (197.421 ± 63.15, P > 0.05. A

  4. Multiple Origins of Plasmodium falciparum Dihydropteroate Synthetase Mutant Alleles Associated with Sulfadoxine Resistance in India▿†

    Science.gov (United States)

    Lumb, Vanshika; Das, Manoj K.; Singh, Neeru; Dev, Vas; Khan, Wajihullah; Sharma, Yagya D.

    2011-01-01

    With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (He) around mutant dhps alleles (He = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean He around the wild-type dhps allele (He = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine- and

  5. Possible role of glutamine synthetase of the prokaryotic type (GSI-like) in nitrogen signaling in Medicago truncatula.

    Science.gov (United States)

    Silva, Liliana S; Seabra, Ana R; Leitão, José N; Carvalho, Helena G

    2015-11-01

    Genes containing domains related to glutamine synthetase of the prokaryotic type (GSI-like) are widespread in higher plants, but their function is currently unknown. To gain insights into the possible role of GSI-like proteins, we characterized the GSI-like gene family of Medicago truncatula and investigated the functionality of the encoded proteins. M. truncatula contains two-expressed GSI-like genes, MtGSIa and MtGSIb, encoding polypeptides of 454 and 453 amino acids, respectively. Heterologous complementation assays of a bacterial glnA mutant indicate that the proteins are not catalytically functional for glutamine synthesis. Gene expression was investigated by qRT-PCR and western blot analysis in different organs of the plant and under different nitrogen (N) regimes, revealing that both genes are preferentially expressed in roots and root nodules, and that their expression is influenced by the N-status of the plant. Analysis of transgenic plants expressing MtGSI-like-promoter-gusA fusion, indicate that the two genes are strongly expressed in the root pericycle, and interestingly, the expression is enhanced at the sites of nodule emergence being particularly strong in specific cells located in front of the protoxylem poles. Taken together, the results presented here support a role of GSI-like proteins in N sensing and/or signaling, probably operating at the interface between perception of the N-status and the developmental processes underlying both root nodule and lateral root formation. This study indicates that GSI-like genes may represent a novel class of molecular players of the N-mediated signaling events. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  6. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins.

    Science.gov (United States)

    Lohman, Jeremy R; Ma, Ming; Cuff, Marianne E; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2014-07-01

    Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs. © 2013 Wiley Periodicals, Inc.

  7. Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

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    Liliana Santos Silva

    2013-09-01

    Full Text Available Nitric oxide (NO is emerging as an important regulatory player in the Rhizobium-legume symbiosis. The occurrence of NO during several steps of the symbiotic interaction suggests an important, but yet unknown, signaling role of this molecule for root nodule formation and functioning. The identification of the molecular targets of NO is key for the assembly of the signal transduction cascade that will ultimately help to unravel NO function. We have recently shown that the key nitrogen assimilatory enzyme Glutamine Synthetase (GS is a molecular target of NO in root nodules of Medicago truncatula, being post-translationally regulated by tyrosine nitration in relation to nitrogen fixation. In functional nodules of M. truncatula NO formation has been located in the bacteroid containing cells of the fixation zone, where the ammonium generated by bacterial nitrogenase is released to the plant cytosol and assimilated into the organic pools by plant GS. We propose that the NO-mediated GS post-translational inactivation is connected to nitrogenase inhibition induced by NO and is related to metabolite channeling to boost the nodule antioxidant defenses. Glutamate, a substrate for GS activity is also the precursor for the synthesis of glutathione (GSH, which is highly abundant in root nodules of several plant species and known to play a major role in the antioxidant defense participating in the ascorbate/GSH cycle. Existing evidence suggests that upon NO-mediated GS inhibition, glutamate could be channeled for the synthesis of GSH. According to this hypothesis, GS would be involved in the NO-signaling responses in root nodules and the NO-signaling events would meet the nodule metabolic pathways to provide an adaptive response to the inhibition of symbiotic nitrogen fixation by reactive nitrogen species (RNS.

  8. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

    Directory of Open Access Journals (Sweden)

    Grigoris D. Amoutzias

    2016-04-01

    Full Text Available Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs and polyketides (PKs are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes and type-I polyketide synthases (PKSes-I, respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  9. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Dang, A.Q.; Faas, F.H.; Carter, W.J.

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  10. Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves.

    Science.gov (United States)

    Lessire, R; Stumpe, P K

    1983-11-01

    Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C(18:0) and C(16:0) were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K(m) values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that beta-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

  11. Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves 1

    Science.gov (United States)

    Lessire, Rene; Stumpe, Paul K.

    1983-01-01

    Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C18:0 and C16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts. The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the Km values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell. PMID:16663268

  12. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    Science.gov (United States)

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. High pretherapeutic thymidylate synthetase and MRP-1 protein levels are associated with nonresponse to neoadjuvant chemotherapy in oesophageal adenocarcinoma patients.

    Science.gov (United States)

    Langer, Rupert; Ott, Katja; Feith, Marcus; Lordick, Florian; Specht, Katja; Becker, Karen; Hofler, Heinz

    2010-10-01

    The aim of this study was to determine whether pretherapeutic protein expression levels of the excision repair cross-complementing (ERCC1) enzyme, thymidylate synthetase (TS), multidrug-resistance protein 1 (MRP-1) and P-glycoprotein (P-gp) are associated with tumour response to cisplatin and fluorouracil (5-FU)-based neoadjuvant chemotherapy in oesophageal adenocarcinomas. The expression levels of ERCC1, TS, MDR-1 and P-gp were determined immunohistochemically in pretherapeutic tumour biopsies from 40 oesophageal adenocarcinoma patients and were correlated with histopathological tumour regression and with patient survival. Protein expression was compared to mRNA data, which was previously published for ERCC1, TS and MRP-1 and newly determined for the purpose of this study for MDR-1/P-gp. High-TS and -MRP-1 protein expression was correlated with tumour non-response to chemotherapy (P = 0.001 and P = 0.036, respectively). For ERCC-1 and P-gp, no association between pretherapeutic protein expression and response was found. There was no correlation between mRNA levels and protein expression for all investigated markers. Survival analysis revealed a trend towards increased survival for low-ERCC-1 expression (P = 0.079). The pattern of pretherapeutic expression of TS and MRP-1 is related to chemotherapy response in oesophageal adenocarcinoma patients. Immunohistochemical assessment of these markers may be helpful for response prediction. J. Surg. Oncol. 2010;102:503-508. © 2010 Wiley-Liss, Inc.

  14. Stage-dependent expression and up-regulation of trypanothione synthetase in amphotericin B resistant Leishmania donovani.

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    Asif Equbal

    Full Text Available Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in Leishmania donovani. In this study, TryS gene of L. donovani (LdTryS was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0-8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of L. major are conserved in L. donovani. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼ 2.0-folds than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H2O2 treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H2O2. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in L. donovani and involvement of TryS during oxidative stress to help the parasites survival.

  15. Elucidating the Pseudomonas aeruginosa fatty acid degradation pathway: identification of additional fatty acyl-CoA synthetase homologues.

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    Jan Zarzycki-Siek

    Full Text Available The fatty acid (FA degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300, which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924 were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617 contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.

  16. A peroxisomal long-chain acyl-CoA synthetase from Glycine max involved in lipid degradation.

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    Lili Yu

    Full Text Available Seed storage oil, in the form of triacylglycerol (TAG, is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525. Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination.

  17. GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light, plant hormones and auxinic herbicides.

    Science.gov (United States)

    Ostrowski, Maciej; Jakubowska, Anna

    2013-03-01

    The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway[S

    OpenAIRE

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

    2013-01-01

    Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-c...

  19. Glutamine Synthetases GLN1;2 and GLN2 in Relation to Arabidopsis Growth Response to Elevated Atmospheric Carbon Dioxide and Varying Nitrogen Forms

    DEFF Research Database (Denmark)

    Vurrakula, Swathi

    content while diluting nitrogen concentrations. Such a reduction in nitrogen concentration will affect plant response to stress and seed/grain yield. Glutamine synthetase (GS) is the central nitrogen-assimilatory enzyme, performing primary and secondary nitrogen assimilation, in response to environmental...... in nitrate assimilation. This was the case as the gln2 mutant biomass was highly reduced with an increased supply of nitrate in solo. Nitrate assimilation was impaired in the mutant in parallel with reduced NR activity and activation state (%), leading to reduced glutamine content while glycine accumulated...

  20. Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex

    DEFF Research Database (Denmark)

    Zou, Zhiying; Tong, Fumin; Færgeman, Nils J.

    2003-01-01

    In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions...... the growth defect in the faa1Delta fat1Delta strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Delta faa1Delta and faa1Delta faa4Delta strains, respectively, indicating Faa1p...

  1. Potentially functional polymorphisms in aminoacyl-tRNA synthetases genes are associated with breast cancer risk in a Chinese population.

    Science.gov (United States)

    He, Yisha; Gong, Jianhang; Wang, Yanru; Qin, Zhenzhen; Jiang, Yue; Ma, Hongxia; Jin, Guangfu; Chen, Jiaping; Hu, Zhibin; Guan, Xiaoxiang; Shen, Hongbing

    2015-07-01

    Aminoacyl-tRNA synthetases (ARSs) are responsible for cellular protein synthesis and cell viability involving in various process of tumorigenesis. We hypothesized that genetic variants in core ARSs genes may play an important role in the development of breast cancer. Thus, we conducted a case-control study including 1064 breast cancer cases and 1073 cancer-free controls to evaluate the associations of 28 potentially functional polymorphisms in 12 core ARSs genes (AARS, CARS, EPRS, HARS, KARS, LARS, MARS, QARS, RARS, VARS, WARS, and YARS) with breast cancer risk. We found significant associations with the risk of breast cancer for rs34087264 in AARS [odds ratio (OR) = 1.15, 95% confidence interval (CI) = 1.01-1.31], rs801186 in HARS (OR = 1.29, 95% CI = 1.08-1.54), rs193466 in RARS (OR = 1.17, 95% CI = 1.02-1.35), and rs2273802 in WARS (OR = 1.14, 95% CI = 1.01-1.30). We further observed significant interactions between rs2273802 and age at the first live birth (P = 0.041), and between rs801186 and age on breast cancer risk (P = 0.018). Combined analysis of these four SNPs showed a significant allele-dosage association between the number of risk alleles and breast cancer risk (Ptrend  = 2.00 × 10(-4) ). Compared with individuals with "0-2" risk alleles, those carrying "3," "4," or "5 or more" risk alleles had a 1.32 (95% CI = 1.07-1.64), 1.48 (95% CI = 1.45-1.91), or 1.60 folds (95% CI = 1.06-2.41) risk of breast cancer, respectively. These findings indicate that genetic variants in core ARSs genes may modify the individual susceptibility to breast cancer in Chinese population. © 2014 Wiley Periodicals, Inc.

  2. Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

    Directory of Open Access Journals (Sweden)

    Tartar Aurélien

    2010-06-01

    Full Text Available Abstract Background Glutamine synthetase (GS is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria to the Chloroplastida. Results GSII sequences were isolated from four species of green algae (Trebouxiophyceae, and additional green algal (Chlorophyceae and Prasinophytae and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB and eukaryotic (GSIIE GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT. Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida. However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting

  3. Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions.

    Science.gov (United States)

    Singh, Kamal Krishna; Ghosh, Shilpi

    2013-02-01

    KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions.

  4. Calcium-mediated responses and glutamine synthetase expression in greater duckweed (Spirodela polyrhiza L.) under diethyl phthalate-induced stress.

    Science.gov (United States)

    Cheng, Lee-Ju; Hung, Meng-Ju; Cheng, Yen-I; Cheng, Tai-Sheng

    2013-11-15

    This study was carried out to assess the influence of diethyl phthalate (DEP) alone or associated with calcium chloride (CaCl2) on greater duckweed plants, emphasizing the implications of calcium in amelioration of DEP-induced stress on plant growth. Greater duckweed were treated with DEP in variable concentrations, as 0, 0.25, 0.5, 1.0 and 2.0mM for 7 days, or treated with the same concentration either 2mM DEP or 2mM DEP plus 10mM CaCl2·2H2O in different duration 0-7 days. Treatment with 2mM DEP resulted in increasing proline content, protease activity, and ammonia accumulation in duckweed tissues. NADH-glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) and Δ(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2), two key enzymes in the glutamate pathway of proline synthesis, showed increase in activity with DEP treatment and positively correlated with proline accumulation. No further increase in proline accumulation was observed with addition of calcium chloride to the DEP-treated cultures. However, supplementation of Ca(2+) can mitigate the adverse effect of DEP, at least in part to decrease the DEP-induced superoxide accumulation and increase in GDH activity for ammonia assimilation in duckweed fronds. In addition, effects of calcium on mitigation of DEP injury were also observed in glutamine synthetase (GS; EC 6.3.1.2) expression. Both GS1 and GS2 polypeptide accumulation and the level of total GS activity were nearly equivalent to the control. Exogenous proline protects GS2 from DEP-modulated redox damage in the chloroplast lysates but there is no remarkable protection effects on D1 (the 32kDa protein in photosystem II reaction center) degradation. In conclusion, the glutamate pathway of proline synthesis might be involved in mitigation of DEP-induced injury, and calcium plays an important role in increasing GDH, P5CR, and GS expression. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Cytosolic glutamine synthetase isozymes play redundant roles in ammonium assimilation under low-ammonium conditions in roots of Arabidopsis thaliana.

    Science.gov (United States)

    Konishi, Noriyuki; Saito, Masahide; Imagawa, Fumi; Kanno, Keiich; Yamaya, Tomoyuki; Kojima, Soichi

    2018-01-24

    Ammonium is a major nitrogen source for plants; it is assimilated into glutamine via a reaction catalyzed by glutamine synthetase (GLN). Arabidopsis expresses four cytosolic GLN genes, GLN1;1, GLN1;2, GLN1;3, and GLN1;4, in roots. However, the function and organization of these GLN1 isozymes in ammonium assimilation in roots remain unclear. In this study, we aimed to characterize the four GLN1 isozymes. The levels of growth of wild type and gln1 single- and multiple-knockout lines were compared in a hydroponic culture at ammonium concentrations of 0.1 and 3 mM. Under the low-ammonium concentration, in single mutants for each GLN1 gene, there was little effect on growth, whereas the triple mutant for GLN1;1, GLN1;2, and GLN1;3 grew slowly and accumulated ammonium. Under the high-ammonium concentration, the single mutant for GLN1;2 showed 50% decreases in fresh weight and glutamine, whereas the other gln1 single mutants did not show notable changes in the phenotype. The double mutant for GLN1;1 and GLN1;2 showed less growth and a lower glutamine concentration than the single mutant for GLN1;2. Promoter analysis indicated an overlapping expression of GLN1;1 with GLN1;2 in the surface layers of the roots. We thus concluded that: 1) at a low concentration, ammonium was assimilated by GLN1;1, GLN1;2, and GLN1;3, and they were redundant; 2) low-affinity GLN1;2 could contribute to ammonium assimilation at concentrations ranging from 0.1 to 3 mM; and 3) GLN1;1 supported GLN1;2 within the outer cell layers of the root. © The Author 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

    Directory of Open Access Journals (Sweden)

    Carter Tamar E

    2012-08-01

    Full Text Available Abstract Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR and sulphadoxine (SDX treatment combination (SP, have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr and dihydropteroate synthetase (dhps genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61 of the samples carried a mutation at codon 108 (S108N of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540 examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59. Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These

  7. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti.

    Science.gov (United States)

    Carter, Tamar E; Warner, Megan; Mulligan, Connie J; Existe, Alexander; Victor, Yves S; Memnon, Gladys; Boncy, Jacques; Oscar, Roland; Fukuda, Mark M; Okech, Bernard A

    2012-08-13

    Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch's T-test p-value = 0.53 and permutations p-value = 0.59). This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on

  8. Identification, expression and IAA-amide synthetase activity analysis of Gretchen Hagen 3 in papaya fruit (Carica papaya L. during postharvest process

    Directory of Open Access Journals (Sweden)

    Kaidong Liu

    2016-10-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  10. Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsu-Hua; Chiang, Yi Ming; Entwistle, Ruth; Ahuja, Mammeet; Lee, Kuan-Han; Bruno, Kenneth S.; Wu, Tung-Kung; Oakley, Berl R.; Wang, Clay C.

    2012-04-10

    Genome sequencing of Aspergillus species including A. nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites have not yet been elucidated. The A. nidulans genome contains twelve nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), and fourteen NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in A. niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

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

    Science.gov (United States)

    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.

  12. The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase.

    Science.gov (United States)

    Cobbett, C S; May, M J; Howden, R; Rolls, B

    1998-10-01

    This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, gamma-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, gamma-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.

  13. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani.

    Science.gov (United States)

    Romans-Fuertes, Patricia; Sondergaard, Teis Esben; Sandmann, Manuela Ilse Helga; Wollenberg, Rasmus Dam; Nielsen, Kristian Fog; Hansen, Frederik T; Giese, Henriette; Brodersen, Ditlev Egeskov; Sørensen, Jens Laurids

    2016-11-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.

  14. Studies on crenarchaeal tyrosylation accuracy with mutational analyses of tyrosyl-tRNA synthetase and tyrosine tRNA from Aeropyrum pernix.

    Science.gov (United States)

    Iwaki, Jun; Endo, Kanako; Ichikawa, Takayuki; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Nishimura, Susumu; Hasegawa, Tsunemi

    2012-12-01

    Aminoacyl-tRNA synthetases play a key role in the translation of genetic code into correct protein sequences. These enzymes recognize cognate amino acids and tRNAs from noncognate counterparts, and catalyze the formation of aminoacyl-tRNAs. While Although several tyrosyl-tRNA synthetases (TyrRSs) from various species have been structurally and functionally well characterized, the crenarchaeal TyrRS remains poorly understood. In this study, we performed mutational analyses on tyrosine tRNA (tRNA(Tyr)) and TyrRS from the crenarchaeon, Aeropyrum pernix, to investigate the molecular recognition mechanism. Kinetics for tyrosylation using in vitro transcript indicated that the discriminator base A73 and adjacent G72 in the acceptor stem are identity elements of tRNA(Tyr), whereas the C1 base and anticodon had modest roles as identity determinants. Intriguingly, in contrast to the identity element of eukaryotic/euryarchaeal TyrRSs, the first base-pair (C1-G72) of the acceptor stem was not essential in crenarchaeal TyrRS as a pair. Furthermore, A. pernix TyrRS mutants were constructed at positions Tyr39 and Asp172, which could form hydrogen bonds with the 4-hydroxyl group of l-tyrosine. The tyrosylation activities with the mutants resulted that Asp172 mutants completely abolished tyrosylation activity, whereas Tyr39 mutants had no effect on activity. Thus, crenarchaeal TyrRS appears to adopt different molecular recognition mechanism from other TyrRSs.

  15. Protein Synthesis During Fungal Spore Germination II. Aminoacyl-soluble Ribonucleic Acid Synthetase Activities During Germination of Botryodiplodia theobromae Spores1

    Science.gov (United States)

    Etten, James L. Van; Brambl, Robert M.

    1968-01-01

    The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores. PMID:5685990

  16. 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.

    Science.gov (United States)

    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.

  17. Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

    Science.gov (United States)

    De Matteis, F.; Gibbs, A.

    1972-01-01

    The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved. PMID:5073727

  18. Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P

    OpenAIRE

    Sumida, Tomomi; Yanagisawa, Tatsuo; Ishii, Ryohei; Yokoyama, Shigeyuki

    2010-01-01

    GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, has been overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant.

  19. Fast dissociation of phe-tRNA synthetase from Aspergillus nidulans immobilized on sepharose-6B column by NaCl.

    Science.gov (United States)

    Singh, N K; Tiwary, B N

    1996-01-01

    Phenylalanyl-tRNA2) synthetase from Aspergillus nidulans was efficiently immobilised to sepharose 6B column containing phenylalanine as the ligand. NaCl was found to be a potent dissociating agent for the immobilised enzyme. While 0.5 M NaCl in discontinuous elution showed a slow impetus on dissociation giving a plateaux profile, a solution of 0.8 M NaCl made the elution rapid giving a sharp peak. On the other hand, in a gradient (continuous) elution the rapidity of dissociation was found to be enhanced with the increase in the difference of the two concentrations. The result suggests that Na+ ions interact with the protein binding site of the ligand eventually dissociating the enzyme molecule by disrupting the covalent bond without affecting its normal catalytic activity.

  20. Novel Hybrid Virtual Screening Protocol Based on Molecular Docking and Structure-Based Pharmacophore for Discovery of Methionyl-tRNA Synthetase Inhibitors as Antibacterial Agents

    Science.gov (United States)

    Liu, Chi; He, Gu; Jiang, Qinglin; Han, Bo; Peng, Cheng

    2013-01-01

    Methione tRNA synthetase (MetRS) is an essential enzyme involved in protein biosynthesis in all living organisms and is a potential antibacterial target. In the current study, the structure-based pharmacophore (SBP)-guided method has been suggested to generate a comprehensive pharmacophore of MetRS based on fourteen crystal structures of MetRS-inhibitor complexes. In this investigation, a hybrid protocol of a virtual screening method, comprised of pharmacophore model-based virtual screening (PBVS), rigid and flexible docking-based virtual screenings (DBVS), is used for retrieving new MetRS inhibitors from commercially available chemical databases. This hybrid virtual screening approach was then applied to screen the Specs (202,408 compounds) database, a structurally diverse chemical database. Fifteen hit compounds were selected from the final hits and shifted to experimental studies. These results may provide important information for further research of novel MetRS inhibitors as antibacterial agents. PMID:23839093

  1. Cytosolic Glutamine Synthetase is Important for Photosynthetic Efficiency and Water Use Efficiency in Potato as Revealed by High Throughput Sequencing QTL analysis

    DEFF Research Database (Denmark)

    Kaminski, Kacper Piotr; Sørensen, Kirsten Kørup; Andersen, Mathias Neumann

    2015-01-01

    efficiency (WUE) of potato is important, but has been limited by technical difficulties in assessing the trait on individual plants and thus is poorly understood. In this study, a mapping population of potatoes has been robustly phenotyped, and considerable variation in WUE under well-watered conditions......Potato (Solanum tuberosum L.) closes its stomata at relatively low soil water deficits frequently encountered in normal field conditions resulting in unnecessary annual yield losses and extensive use of artificial irrigation. Therefore, unraveling the genetics underpinning variation in water use...... isoforms of cytosolic glutamine synthase were located in the QTL at chromosome 1 suggesting a major contribution of this enzyme to photosynthetic efficiency and thus WUE in potato. Indeed, Glutamine synthetase enzyme activity of leaf extracts was measured and found to be correlated with contrasting WUE...

  2. From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.

    Science.gov (United States)

    Gaca, Anthony O; Kudrin, Pavel; Colomer-Winter, Cristina; Beljantseva, Jelena; Liu, Kuanqing; Anderson, Brent; Wang, Jue D; Rejman, Dominik; Potrykus, Katarzyna; Cashel, Michael; Hauryliuk, Vasili; Lemos, José A

    2015-09-01

    The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing

  3. In silico analysis highlights the frequency and diversity of type 1 lantibiotic gene clusters in genome sequenced bacteria

    LENUS (Irish Health Repository)

    Marsh, Alan J

    2010-11-30

    Abstract Background Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria. Results In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production. Conclusions Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.

  4. A designated centre for people with disabilities operated by COPE Foundation - Cork

    LENUS (Irish Health Repository)

    Marsh, Alan J

    2010-11-30

    Abstract Background Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria. Results In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production. Conclusions Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.

  5. A designated centre for people with disabilities operated by Ability West, Galway

    LENUS (Irish Health Repository)

    Marsh, Alan J

    2010-11-30

    Abstract Background Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria. Results In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production. Conclusions Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.

  6. The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a).

    Science.gov (United States)

    Molina-Rueda, Juan Jesús; Tsai, Chung Jui; Kirby, Edward G

    2013-01-01

    Glutamine synthetase (GS) plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4) expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a) display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD) family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs). The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs), with concomitant up-regulation of plastidic iron SODs (FSDs) in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated down-regulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this copper-mediated mechanism represents a common

  7. The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection.

    Directory of Open Access Journals (Sweden)

    Marcell Crispim

    2018-01-01

    Full Text Available Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

  8. Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence.

    Science.gov (United States)

    Orsel, Mathilde; Moison, Michaël; Clouet, Vanessa; Thomas, Justine; Leprince, Françoise; Canoy, Anne-Sophie; Just, Jérémy; Chalhoub, Boulos; Masclaux-Daubresse, Céline

    2014-07-01

    A total of 16 BnaGLN1 genes coding for cytosolic glutamine synthetase isoforms (EC 6.3.1.2.) were found in the Brassica napus genome. The total number of BnaGLN1 genes, their phylogenetic relationships, and genetic locations are in agreement with the evolutionary history of Brassica species. Two BnaGLN1.1, two BnaGLN1.2, six BnaGLN1.3, four BnaGLN1.4, and two BnaGLN1.5 genes were found and named according to the standardized nomenclature for the Brassica genus. Gene expression showed conserved responses to nitrogen availability and leaf senescence among the Brassiceae tribe. The BnaGLN1.1 and BnaGLN1.4 families are overexpressed during leaf senescence and in response to nitrogen limitation. The BnaGLN1.2 family is up-regulated under high nitrogen regimes. The members of the BnaGLN1.3 family are not affected by nitrogen availability and are more expressed in stems than in leaves. Expression of the two BnaGLN1.5 genes is almost undetectable in vegetative tissues. Regulations arising from plant interactions with their environment (such as nitrogen resources), final architecture, and therefore sink-source relations in planta, seem to be globally conserved between Arabidopsis and B. napus. Similarities of the coding sequence (CDS) and protein sequences, expression profiles, response to nitrogen availability, and ageing suggest that the roles of the different GLN1 families have been conserved among the Brassiceae tribe. These findings are encouraging the transfer of knowledge from the Arabidopsis model plant to the B. napus crop plant. They are of special interest when considering the role of glutamine synthetase in crop yield and grain quality in maize and wheat. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance.

    Science.gov (United States)

    Lim, B; Pasternak, M; Meyer, A J; Cobbett, C S

    2014-01-01

    The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2 O2 ) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  10. The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a.

    Directory of Open Access Journals (Sweden)

    Juan Jesús Molina-Rueda

    Full Text Available BACKGROUND: Glutamine synthetase (GS plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4 expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. RESULTS: In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs. The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs, with concomitant up-regulation of plastidic iron SODs (FSDs in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated down-regulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. CONCLUSIONS: Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this

  11. Calcium-induced proline accumulation contributes to amelioration of NaCl injury and expression of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

    Science.gov (United States)

    Cheng, Tai-Sheng; Hung, Meng-Ju; Cheng, Yen-I; Cheng, Lee-Ju

    2013-11-15

    The calcium-mediated proline accumulation is a critical response under NaCl stress and the function of the induced proline as a glutamine synthetase (GS) protectant in greater duckweed was investigated. The plants were treated with solutions containing 100mM NaCl, 200 mM NaCl, 200 mM NaCl plus 10mM CaCl2, or 10mM CaCl2 alone for 4 days. At the end of the experiment, the fronds of inoculum treated with 200 mM NaCl showed the chlorotic effect, higher glutamate dehydrogenase (NADH-GDH) activity and lower GS activity. At the lower salinity, the activities of GS and NADH-GDH were not altered markedly. A significant accumulation of proline was not found under either low or high salinity. The activity of Δ(1)-pyrroline-5-carboxylate reductase (P5CR) was enhanced only at 200 mM NaCl but remained unchanged at 100mM NaCl. The activity of Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) did not change under salinity-stressed. Addition of CaCl2 to the salt stressed plants not only lowered NaCl injury but also showed an elevated level of proline contents in response to the salinity treatment. In addition, both GS activity and corresponding polypeptides were expressed close to the level of control. Exogenous proline protects GS2 and the 32 kDa protein in photosystem II reaction center (D1) from H2O2-induced redox degradation in the chloroplast lysates of duckweed. The results suggest that calcium-induced proline accumulation may play an important role as a GS protectant under NaCl exposure in S. polyrhiza. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145

    Directory of Open Access Journals (Sweden)

    Agnieszka Bera

    2017-04-01

    Full Text Available Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962, was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.

  13. Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

    Science.gov (United States)

    Dahlquist, F W; Purich, D L

    1975-05-06

    The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.

  14. Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

    Directory of Open Access Journals (Sweden)

    Wagner Martin

    2006-03-01

    Full Text Available Abstract Background Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS, but its exact genetic organization and biochemical synthesis is unknown. Results The complete sequence of the cereulide synthetase (ces gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. Conclusion The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces

  15. Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-02-01

    Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).

  16. Effect of continuous-wave and amplitude-modulated 2.45 GHz microwave radiation on the liver and brain aminoacyl-transfer RNA synthetases of in utero exposed mice

    Energy Technology Data Exchange (ETDEWEB)

    Kubinyi, G.; Thuroczy, G.; Bakos, J.; Boeloeni, E.; Sinay, H.; Szabo, L.D. [National Frederic Joliot-Curie Research Inst. for Radiobiology and Radiohygiene, Budapest (Hungary)

    1996-12-31

    Investigations have been carried out concerning the effects of microwave (MW) exposure on the aminoacyl-transfer ribonucleic acid (tRNA) synthetase of the progeny of females that were exposed during their entire period of gestation (19 days). The changes caused by continuous-wave (CW) and amplitude-modulated (AM) MW radiation have been compared. CFLP mice were exposed to MW radiation for 100 min each day in an anechoic room. The MW frequency was 2.45 GHz, and the amplitude modulation had a 50 Hz rectangular waveform (on/off ratio, 50/50%). The average power density exposure was 3 mW/cm{sup 2}, and the whole body specific absorption rate (SAR) was 4.23 {+-} 0.63 W/kg. The weight and mortality of the progeny were followed until postnatal day 24. Aminoacyl-tRNA synthetase enzymes and tRNA from the brains and livers of the offspring (461 exposed, 487 control) were isolated. The aminoacyl-tRNA synthetase activities were determined. The postnatal increase of body weight and organ weight was not influenced by the prenatal MW radiation. The activity of enzyme isolated form the brain showed a significant decrease after CW MW exposure, but the changes were not significant after 50 Hz AM MW exposure. The activity of the enzyme isolated from liver increased under CW and 50 Hz modulated MW.

  17. Molecular modeling of the reductase domain to elucidate the reaction mechanism of reduction of peptidyl thioester into its corresponding alcohol in non-ribosomal peptide synthetases

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    Lee Gwang

    2010-01-01

    Full Text Available Abstract Background Nonribosomal peptide synthetases (NRPSs are multienzymatic, multidomain megasynthases involved in the biosynthesis of pharmaceutically important nonribosomal peptides. The peptaibol synthetase from Trichoderma virens (TPS is an important member of the NRPS family that exhibits antifungal properties. The majority of the NRPSs terminate peptide synthesis with the thioesterase (TE domain, which either hydrolyzes the thioester linkage, releasing the free peptic acid, or catalyzes the intramolecular macrocyclization to produce a macrolactone product. TPS is an important NRPS that does not encompass a TE domain, but rather a reductase domain (R domain to release the mature peptide product reductively with the aid of a NADPH cofactor. However, the catalytic mechanism of the reductase domain has not yet been elucidated. Results We present here a three-dimensional (3D model of the reductase domain based on the crystal structure of vestitone reductase (VR. VR belongs to the short-chain dehydrogenase/reductase (SDR superfamily and is responsible for the nicotinamide dinucleotide phosphate (NADPH-dependent reduction of the substrate into its corresponding secondary alcohol product. The binding sites of the probable linear substrates, alamethicin, trichotoxin, antiamoebin I, chrysopermin C and gramicidin, were identified within the modeled R domain using multiple docking approaches. The docking results of the ligand in the active site of the R domain showed that reductase side chains have a high affinity towards ligand binding, while the thioester oxygen of each substrate forms a hydrogen bond with the OH group of Tyr176 and the thiol group of the substrate is closer to the Glu220. The modeling and docking studies revealed the reaction mechanism of reduction of thioester into a primary alcohol. Conclusion Peptaibol biosynthesis incorporates a single R domain, which appears to catalyze the four-electron reduction reaction of a peptidyl

  18. Molecular modeling and molecular dynamics simulation study of archaeal leucyl-tRNA synthetase in complex with different mischarged tRNA in editing conformation.

    Science.gov (United States)

    Rayevsky, A V; Sharifi, M; Tukalo, M A

    2017-09-01

    Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNALeu was modeled wherever tRNA's acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of PhLeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by PhLeuRS's editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNALeu and isoleucyl-tRNALeu. In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by PhLeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS (TtLeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of PhLeuRS differs from that of prokaryotic TtLeuRS. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products

    Science.gov (United States)

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to ...

  20. Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4).

    Science.gov (United States)

    Coleman, Heather D; Cánovas, Francisco M; Man, Huimin; Kirby, Edward G; Mansfield, Shawn D

    2012-09-01

    Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3-year-old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild-type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid-insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild-type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  1. Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol.

    Science.gov (United States)

    Hartmann, Tanja; Hönicke, Petra; Wirtz, Markus; Hell, Rüdiger; Rennenberg, Heinz; Kopriva, Stanislav

    2004-04-01

    Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.

  2. Characterization of benzoxaborole-based antifungal resistance mutations demonstrates that editing depends on electrostatic stabilization of the leucyl-tRNA synthetase editing cap.

    Science.gov (United States)

    Sarkar, Jaya; Mao, Weimin; Lincecum, Tommie L; Alley, M R K; Martinis, Susan A

    2011-10-03

    The broad-spectrum benzoxaborole antifungal AN2690 blocks protein synthesis by inhibiting leucyl-tRNA synthetase (LeuRS) via a novel oxaborole tRNA trapping mechanism in the editing site. Herein, one set of resistance mutations is at Asp487 outside the LeuRS hydrolytic editing pocket, in a region of unknown function. It is located within a eukaryote/archaea specific insert I4, which forms part of a cap over a benzoxaborole-AMP that is bound in the LeuRS CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap of yeast LeuRS that is critical for tRNA deacylation. We hypothesize that this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Adaptive Calcified Matrix Response of Dental Pulp to Bacterial Invasion Is Associated with Establishment of a Network of Glial Fibrillary Acidic Protein+/Glutamine Synthetase+ Cells

    Science.gov (United States)

    Farahani, Ramin M.; Nguyen, Ky-Anh; Simonian, Mary; Hunter, Neil

    2010-01-01

    We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein− cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP+/GS+ cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP+/GS+ interodontoblastic cells. A regulatory role for the networks of GFAP+/GS+ cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response. PMID:20802180

  4. IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

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    Jand-Venes Rolim MEDEIROS

    2013-04-01

    Full Text Available Context Hydrogen sulphide (H2S has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. Objective To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE and cystathionine-β-synthetase (CBS isoforms in gastric mucosa of mice treated with saline or 50% ethanol. Methods Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g. After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. Results We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. Conclusion Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

  5. The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1)) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine.

    Science.gov (United States)

    Simon, Bindu; Sengupta-Gopalan, Champa

    2010-10-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

  6. Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats.

    Science.gov (United States)

    Cai, Cheng; Qiu, Gang; Gong, Xiaohui; Chen, Yihuan; Zhao, Huanhu

    2014-01-01

    To explore the effect of erythromycin on hyperoxia-induced lung injury. One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  7. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

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    Mykuliak V. V.

    2014-03-01

    Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

  8. 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.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

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

    2015-06-01

    Here, we investigated the roles of YAL1 (FAA1) and FAT1 encoding acyl-CoA synthetases (ACSs) and three additional orthologs of ACS genes FAT2-FAT4 of the yeast Yarrowia lipolytica in the assimilation or utilization of n-alkanes and fatty acids. ACS deletion mutants were generated to characterize their function. The FAT1 deletion mutant exhibited decreased growth on n-alkanes of 10-18 carbons, whereas the FAA1 mutant showed growth reduction on n-alkane of 16 carbons. However, FAT2-FAT4 deletion mutants did not show any growth defects, suggesting that FAT1 and FAA1 are involved in the activation of fatty acids produced during the metabolism of n-alkanes. In contrast, deletions of FAA1 and FAT1-FAT4 conferred no defect in growth on fatty acids. The wild-type strain grew in the presence of cerulenin, an inhibitor of fatty acid synthesis, by utilizing exogenously added fatty acid or fatty acid derived from n-alkane when oleic acid or n-alkane of 18 carbons was supplemented. However, the FAA1 deletion mutant did not grow, indicating a critical role for FAA1 in the utilization of fatty acids. Fluorescent microscopic observation and biochemical analyses suggested that Fat1p is present in the peroxisome and Faa1p is localized in the cytosol and to membranes. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

    Science.gov (United States)

    Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

    2017-01-27

    The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

    Science.gov (United States)

    Nam, Seo Hee; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Kim, Doyeun; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Sunghoon; Lee, Jung Weon

    2016-04-01

    The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

  12. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions.

    Science.gov (United States)

    Hong, Hye-Jin; Lim, Hui Xuan; Song, Ju Han; Lee, Arim; Kim, Eugene; Cho, Daeho; Cohen, Edward P; Kim, Tae Sung

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

  13. Light and metabolic regulation of HAS1, HAS1.1 and HAS2, three asparagine synthetase genes in Helianthus annuus.

    Science.gov (United States)

    Herrera-Rodríguez, María Begoña; Maldonado, José María; Pérez-Vicente, Rafael

    2004-06-01

    The role of light, carbon and nitrogen availability on the regulation of three asparagine synthetase (AS, EC 6.3.5.4)-coding genes, HAS1, HAS1.1 and HAS2, has been investigated in sunflower (Helianthus annuus). The response of each gene to different illumination conditions and to treatments that modify the carbon and nitrogen status of the plant was evaluated by Northern analysis with gene-specific probes. Light represses the expression of HAS1 and HAS1.1. Phytochrome and photosynthesis-derived carbohydrates mediate this repression. On the contrary, maintained HAS2 expression requires light and is positively affected by sucrose. HAS1 and HAS1.1 expression is dependent on nitrogen availability, while HAS2 transcripts are still found in N-starved plants. High ammonium level induces all three AS genes and partially reverts sucrose repression of HAS1 and HAS1.1. In summary, light, carbon and nitrogen availability control asparagine synthesis in sunflower by regulating three AS-coding genes. Illumination and carbon sufficiency maintain HAS2 active to supply asparagine that can be used for growth. Darkness and low C/N ratio conditions trigger the response of the specialized HAS1 and HAS1.1 genes which contribute to store the excess nitrogen as asparagine. Ammonium induces all three AS-genes which may favor its detoxification.

  14. Mutation of the Mitochondrial Tyrosyl-tRNA Synthetase Gene, YARS2, Causes Myopathy, Lactic Acidosis, and Sideroblastic Anemia—MLASA Syndrome

    Science.gov (United States)

    Riley, Lisa G.; Cooper, Sandra; Hickey, Peter; Rudinger-Thirion, Joëlle; McKenzie, Matthew; Compton, Alison; Lim, Sze Chern; Thorburn, David; Ryan, Michael T.; Giegé, Richard; Bahlo, Melanie; Christodoulou, John

    2010-01-01

    Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA. PMID:20598274

  15. X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture.

    Science.gov (United States)

    Tarry, Michael J; Haque, Asfarul S; Bui, Khanh Huy; Schmeing, T Martin

    2017-05-02

    Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Value and Limits of Routine Histology Alone or Combined with Glutamine Synthetase Immunostaining in the Diagnosis of Hepatocellular Adenoma Subtypes on Surgical Specimens

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    Paulette Bioulac-Sage

    2013-01-01

    Full Text Available Immunohistochemistry is a valid method to classify hepatocellular adenoma (HCA. The aim was to test the performance of routine histology combined to glutamine synthetase (GS staining to identify the 2 major HCA subtypes: HNF1α inactivated (H-HCA and inflammatory HCA (IHCA. 114 surgical cases, previously classified by immunohistochemistry, were analysed. Group A comprised 45 H-HCAs, 44 IHCAs, and 9 β-catenin-activated IHCAs (b-IHCA, and group B, 16 b-HCA and unclassified HCA (UHCA. Steatosis was the hallmark of H-HCA. IHCA and b-IHCA were mainly characterized by inflammation, thick arteries, and sinusoidal dilatation; b-IHCA could not be differentiated from IHCA by routine histology. Group B was identified by default. A control set (91 cases was analyzed using routine and GS stainings (without knowing immunohistochemical results. Among the 45 H-HCAs and 27 IHCAs, 40 and 24 were correctly classified, respectively. Among the 10 b-IHCAs, 4 were identified as such using additional GS. Eight of the 9 HCAs that were neither H-HCA nor IHCA were correctly classified. Conclusion. Routine histology allows to diagnose >85% of the 2 major HCA subtypes. GS is essential to identify b-HCA. This study demonstrates that a “palliative” diagnostic approach can be proposed, when the panel of specific antibodies is not available.

  17. Acetate formation in the photoheterotrophic bacterium Chloroflexus aurantiacus involves an archaeal type ADP-forming acetyl-CoA synthetase isoenzyme I.

    Science.gov (United States)

    Schmidt, Marcel; Schönheit, Peter

    2013-12-01

    The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria, could not be detected; instead, the cells contained an acetyl-CoA synthetase (ADP-forming) (ACD) (acetyl-CoA + ADP + Pi → acetate + ATP + CoA), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea. ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl-CoA/acetate and other acyl-CoA esters/acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. Influence of the temporal and spatial variation of nitrate reductase, glutamine synthetase and soil composition in the N species content in lettuce (Lactuca sativa).

    Science.gov (United States)

    Pinto, Edgar; Fidalgo, Fernanda; Teixeira, Jorge; Aguiar, Ana A; Ferreira, Isabel M P L V O

    2014-04-01

    The variation of nitrate reductase (NR), glutamine synthetase (GS) and N content in lettuce was evaluated at 5 stages of lettuce growth. Soil physicochemical properties and its N content were also assessed to elucidate the soil-to-plant transfer of inorganic N and potential leaching to groundwater. A decrease of NR activity and an increase of NO3(-) and N-Kjeldahl content in lettuces were observed during plant growth, whereas GS activity and NH4(+) increased during the first few weeks of lettuce growth and then decreased. Although the temporal variation was similar in lettuces grown in different soils, quantitative differences were observed, indicating that high NO3(-) content in soil caused a higher NO3(-) accumulation in lettuce despite the higher NR activity during the initial stage of plant growth. Higher levels of NO3(-) and NH4(+) were correlated with higher levels of N-Kjeldahl in lettuce suggesting a positive effect of these N species in the biosynthesis of organic forms of N. Soil physicochemical properties influenced the mobility of inorganic N within the groundwater-soil-plant system. Sandy soils with low OM content allowed NO3(-) leaching, which was confirmed by higher NO3(-) levels in groundwater. Therefore, lettuces grown in those soils presented lower N content and the inputs of N to the environment were higher. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. The Stable Level of Glutamine synthetase 2 Plays an Important Role in Rice Growth and in Carbon-Nitrogen Metabolic Balance

    Science.gov (United States)

    Bao, Aili; Zhao, Zhuqing; Ding, Guangda; Shi, Lei; Xu, Fangsen; Cai, Hongmei

    2015-01-01

    Glutamine synthetase 2 (GS2) is a key enzyme involved in the ammonium metabolism in plant leaves. In our previous study, we obtained GS2-cosuppressed plants, which displayed a normal growth phenotype at the seedling stage, while at the tillering stage they showed a chlorosis phenotype. In this study, to investigate the chlorosis mechanism, we systematically analyzed the plant growth, carbon-nitrogen metabolism and gene expressions between the GS2-cosuppressed rice and wild-type plants. The results revealed that the GS2-cosuppressed plants exhibited a poor plant growth phenotype and a poor nitrogen transport ability, which led to nitrogen accumulation and a decline in the carbon/nitrogen ratio in the stems. Interestingly, there was a higher concentration of soluble proteins and a lower concentration of carbohydrates in the GS2-cosuppressed plants at the seedling stage, while a contrasting result was displayed at the tillering stage. The analysis of the metabolic profile showed a significant increase of sugars and organic acids. Additionally, gene expression patterns were different in root and leaf of GS2-cosuppressed plants between the seedling and tillering stage. These results indicated the important role of a stable level of GS2 transcription during normal rice development and the importance of the carbon-nitrogen metabolic balance in rice growth. PMID:26053400

  20. Effect of Electroacupuncture on the Expression of Glycyl-tRNA Synthetase and Ultrastructure Changes in Atrophied Rat Peroneus Longus Muscle Induced by Sciatic Nerve Injection Injury

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    Meng Wang

    2016-01-01

    Full Text Available Glycyl-tRNA synthetase (GlyRS is one of the key enzymes involved in protein synthesis. Its mutations have been reported to cause Charcot-Marie-Tooth disease which demonstrates muscular atrophy in distal extremities, particularly manifested in peroneus muscles. In this situation, the dysfunctions of mitochondria and sarcoplasmic reticulum (SR affect energy supply and excitation-contraction coupling of muscle fibers, therefore resulting in muscular atrophy. Although the treatment of muscular atrophy is a global urgent problem, it can be improved by electroacupuncture (EA treatment. To investigate the mechanism underlying EA treatment improving muscular atrophy, we focused on the perspective of protein synthesis by establishing a penicillin injection-induced sciatic nerve injury model. In our model, injured rats without treatment showed decreased sciatic functional index (SFI, decreased peroneus longus muscle weight and muscle fiber cross-sectional area, aggregated mitochondria with vacuoles appearing, swollen SR, and downregulated mRNA and protein expression levels of GlyRS and myosin heavy chain IIb (MHC-IIb. The injured rats with EA treatment showed significant recovery. These results indicated that EA stimulation can alleviate peroneus longus muscular atrophy induced by iatrogenic sciatic nerve injury through promoting the recovery of GlyRS and muscle ultrastructure and increasing muscle protein synthesis.

  1. A high-throughput absorbance-based assay for methionine produced by methionine aminopeptidase using S-adenosyl-L-methionine synthetase.

    Science.gov (United States)

    Shapiro, Adam B; Gao, Ning; Thresher, Jason; Walkup, Grant K; Whiteaker, James

    2011-06-01

    Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.

  2. Transplantation of N-Acetyl Aspartyl-Glutamate Synthetase-Activated Neural Stem Cells after Experimental Traumatic Brain Injury Significantly Improves Neurological Recovery

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    Mingfeng Li

    2013-12-01

    Full Text Available Background/Aims: Neural stem cells (NSCs hold considerable potential as a therapeutic tool for repair of the damaged nervous system. In the current study, we examined whether transplanted N-acetyl aspartyl-glutamate synthetase (NAAGS-activated NSCs (NAAGS/NSCs further improve neurological recovery following traumatic brain injury (TBI in Sprague-Dawley rats. Methods: Animals received TBI and stereotactic injection of NSCs, NAAGS/NSCs or phosphate buffered saline without cells (control into the injured cortex. NAAGS protein expression was detected through western blot analysis. Dialysate NAAG levels were analyzed with radioimmunoassay. Cell apoptosis was detected via TUNEL staining. The expression levels of specific pro-inflammatory cytokines were detected with enzyme-linked immunosorbent assay. Results: Groups with transplanted NSCs and NAAGS/NSCs displayed significant recovery of the motor behavior, compared to the control group. At 14 and 21 days post-transplantation, the motor behavior in NAAGS/NSC group was significantly improved than that in NSC group (pConclusion: Our results collectively demonstrate that NAAGS/NSCs provide a more powerful autoplastic therapy for the injured nervous system.

  3. Acyl coenzyme A synthetase long-chain 1 (ACSL1 gene polymorphism (rs6552828 and elite endurance athletic status: a replication study.

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    Thomas Yvert

    Full Text Available The aim of this study was to determine the association between the rs6552828 polymorphism in acyl coenzyme A synthetase (ACSL1 and elite endurance athletic status. We studied 82 Caucasian (Spanish World/Olympic-class endurance male athletes, and a group of sex and ethnically matched healthy young adults (controls, n=197. The analyses were replicated in a cohort of a different ethnic origin (Chinese of the Han ethnic group, composed of elite endurance athletes (runners [cases, n=241 (128 male] and healthy sedentary adults [controls, n=504 (267 male]. In the Spanish cohort, genotype (P=0.591 and minor allele (A frequencies were similar in cases and controls (P=0.978. In the Chinese cohort, genotype (P=0.973 and minor allele (G frequencies were comparable in female endurance athletes and sedentary controls (P=0.881, whereas in males the frequency of the G allele was higher in endurance athletes (0.40 compared with their controls (0.32, P=0.040. The odds ratio (95%CI for an elite endurance Chinese athlete to carry the G allele compared with ethnically matched controls was 1.381 (1.015-1.880 (P-value=0.04. Our findings suggest that the ACSL1 gene polymorphism rs6552828 is not associated with elite endurance athletic status in Caucasians, yet a marginal association seems to exist for the Chinese (Han male population.

  4. Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice.

    Science.gov (United States)

    Ding, Xinhua; Cao, Yinglong; Huang, Liling; Zhao, Jing; Xu, Caiguo; Li, Xianghua; Wang, Shiping

    2008-01-01

    New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.

  5. Thioredoxin-Interacting Protein (TXNIP) Suppresses Expression of Glutamine Synthetase by Inducing Oxidative Stress in Retinal Muller Glia Under Diabetic Conditions.

    Science.gov (United States)

    Zhou, Jia; Shen, Xi; Lu, Qiong; Zhang, Min

    2016-05-01

    BACKGROUND Diabetic retinopathy (DR) is a progressive neurodegenerative disease with early-stage symptoms such as dysfunction of Muller cells, which leads to ganglion cell death. Its pathogenesis is probably associated with oxidative stress and a recently discovered protein, thioredoxin-interacting protein (TXNIP). MATERIAL AND METHODS To explore the role of TXNIP in DR, we cultured Muller cells under diabetic conditions, and then used immunohistochemistry, Western blot, and RT-PCR to detect the expression level of TXNIP under diabetic conditions. We demonstrated the expression level of glutamine synthetase (GS) when TXNIP was inhibited. To explore the potential pathway of TXNIP-induced cell damage in DR, we confirmed the role of IL-1β under diabetic conditions. RESULTS Diabetes induces TXNIP expressions at mRNA levels, but shows the opposite effect on GS. IL-1β plays an important role in this pathway. Azaserine effectively increased the expression of GS via attenuating the expression of TXNIP. CONCLUSIONS This study demonstrates the role of TXNIP and its mechanism in DR, provides a possible treatment for DR, and lays a new theoretical foundation for the clinical treatment of DR and other diabetic microvascular changes.

  6. Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells

    Science.gov (United States)

    Wang, Lina; Lin, Ye; Bian, Yanjie; Liu, Lili; Shao, Li; Lin, Lin; Qu, Bo; Zhao, Feng; Gao, Xuejun; Li, Qingzhang

    2014-01-01

    The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration. PMID:24722568

  7. Paternal retrieval behavior regulated by brain estrogen synthetase (aromatase in mouse sires that engage in communicative interactions with pairmates

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    Shirin eAkther

    2015-12-01

    Full Text Available Parental behaviors involve complex social recognition and memory processes and interactive behavior with children that can greatly facilitate healthy human family life. Fathers play a substantial role in child care in a small but significant number of mammals, including humans. However, the brain mechanism that controls male parental behavior is much less understood than that controlling female parental behavior. Fathers of non-monogamous laboratory ICR mice are an interesting model for examining the factors that influence paternal responsiveness because sires can exhibit maternal-like parental care (retrieval of pups when separated from their pups along with their pairmates because of olfactory and auditory signals from the dams. Here we tested whether paternal behavior is related to femininity by the aromatization of testosterone. For this purpose, we measured the immunoreactivity of aromatase (cytochrome P450 family 19 (CYP19, which synthesizes estrogen from androgen, in nine brain regions of the sire. We observed higher levels of aromatase expression in these areas of the sire brain when they engaged in communicative interactions with dams in separate cages. The capacity of sires to retrieve pups was increased following a period of five days spent with the pups as a whole family after parturition, whereas the acquisition of this ability was suppressed in sires treated daily with an aromatase inhibitor. These results suggest that brain aromatization regulates the initiation, development, and maintenance of paternal behavior in the ICR mice.

  8. Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress

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    Chunquan Ma

    2017-04-01

    Full Text Available The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH. A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS, is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM, a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H2O2 tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet.

  9. Characterization of TLR4-mediated auto-antibody production in a mouse model of histidyl-tRNA synthetase-induced myositis.

    Science.gov (United States)

    Harlow, Lisa; Fernandez, Irina; Soejima, Makoto; Ridgway, William M; Ascherman, Dana P

    2012-12-01

    We have previously shown that intramuscular immunization with a recombinant fragment of murine histidyl-tRNA synthetase (HRS) in the absence of exogenous adjuvant generates Ag-specific, IgG class switched Abs a murine model of myositis. Markedly diminished IgG anti-HRS auto-Ab responses in TLR4 signaling-deficient C3H/HeJ mice indicate that TLR4 is required for auto-Ab formation and/or class switching in this system. Comparative time course assessment of HRS-immunized C3H/HeOuJ (wild type) and C3H/HeJ (TLR4 mutant) mice shows here that despite significant impairment of class switched IgG anti-HRS responses in TLR4-deficient C3H/HeJ mice, production of IgM anti-HRS auto-Abs is relatively preserved-suggesting that TLR4-mediated signals modulate IgG class switching rather than auto-Ab formation in this genetic background. In C57BL/6-derived knockout mice lacking either MyD88 (B6.MyD88(-/-)) or TRIF (B6.TRIF(-/-)) adaptor molecules, immunization studies indicate that TRIF exerts a dominant role in the generation of HRS-specific IgG auto-Abs. Complementing these analyses, in vitro stimulation of unfractionated, as well as T cell-depleted, C3H/HeOuJ splenocytes with recombinant murine HRS reveals that TLR4-mediated generation of class switched auto-Abs can occur independently of T cell help. Overall, these findings support a broader role for TLR4 in the breakdown of immune tolerance and development of autoimmunity.

  10. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

    Science.gov (United States)

    2010-01-01

    Background Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. Results CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. Conclusions The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of

  11. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

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    Lovley Derek R

    2010-07-01

    Full Text Available Abstract Background Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. Results CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. Conclusions The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of

  12. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway.

    Science.gov (United States)

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

    2013-05-01

    Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

  13. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway[S

    Science.gov (United States)

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

    2013-01-01

    Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis. PMID:23427282

  14. Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

    Science.gov (United States)

    Takatani, Nobuyuki; Use, Kazuhide; Kato, Akihiro; Ikeda, Kazutaka; Kojima, Kouji; Aichi, Makiko; Maeda, Shin-Ichi; Omata, Tatsuo

    2015-08-01

    Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms.

    Science.gov (United States)

    Lo, Jun-Chih; Huang, Wei-Chi; Chou, Yun-Chia; Tseng, Chun-Hsien; Lee, Wei-Li; Sun, Synthia H

    2008-04-01

    Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.

  16. Kinetic evidence for half-of-the-sites reactivity in tRNA/sup Trp/ aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

    Energy Technology Data Exchange (ETDEWEB)

    Trezeguet, V.; Merle, M.; Gandar, J.C.; Labouesse, B.

    1986-11-04

    The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flowed method. The transfer of tryptophan to tRNA/sup Trp/ was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-(/sup 14/C)tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-(/sup 14/C)tryptophan were used. The authors measured either the formation of a single labeled aminoacyl-tRNA/sup Trp/ per enzyme subunit or the turnover of labeled aminoacyl-tRNA/sup Trp/ synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute half-of-the-sites reactivity); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNA/sup Trp/ under each labeling conditions were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate if aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.

  17. The rainbow trout CMP-sialic acid synthetase utilises a nuclear localization signal different from that identified in the mouse enzyme.

    Science.gov (United States)

    Tiralongo, Joe; Fujita, Akiko; Sato, Chihiro; Kitajima, Ken; Lehmann, Friederike; Oschlies, Melanie; Gerardy-Schahn, Rita; Münster-Kühnel, Anja K

    2007-09-01

    The terminal sugar sialic acid (Sia) plays a pivotal role in cell-cell interaction and recognition. A prerequisite for the biosynthesis of sialoglycoconjugates is the activation of Sia to cytidine monophosphate-Sia (CMP-Sia), by CMP-Sia synthetases (CMP-Sia-syn). CMP-Sia-syn are conserved from bacteria to man, and have been found to reside in the nucleus of all vertebrate species analysed to date. We previously cloned the CMP-Sia-syn from rainbow trout (rt) and identified three clusters of basic amino acids (BC) that might act as nuclear localization signals (NLS). Here, we utilised chimeric proteins and rt CMP-Sia-syn mutants in which putative NLS sequences were deleted, to identify the nuclear transport signal. Divergent from the mouse enzyme, where the crucial NLS is part of the enzyme's active site, in the rt CMP-Sia-syn the NLS and active site are disparate. The crucial NLS in the fish enzyme is bipartite and the functionality depends on a free N-terminus. Comparative analysis of all putative rt NLS in mouse and fish cells identified a second inferior motif (rtBC5-6), which was functional only in fish cells suggesting some differences in transport mechanism or folding variabilities in fish. Moreover, based on computational analyses of putative CMP-Sia-syn from distant deuterostomian organisms it was concluded that CMP-Sia-syn nuclear localization is a relatively recent invention, originating in echinoderms. In summary, our data describing structural differences in the NLS of vertebrate CMP-Sia-syn, and the independence of Sia activation from the subcellular localization of the enzyme, provide supporting evidence that nuclear localization is linked to a second yet unknown function.

  18. Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases.

    Science.gov (United States)

    Heyes, Derren J; Levy, Colin; Lafite, Pierre; Roberts, Ian S; Goldrick, Marie; Stachulski, Andrew V; Rossington, Steven B; Stanford, Deborah; Rigby, Stephen E J; Scrutton, Nigel S; Leys, David

    2009-12-18

    The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

  19. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

  20. Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits

    Directory of Open Access Journals (Sweden)

    Edit Németh

    2018-02-01

    Full Text Available Glutamine synthetase (GS is of central interest as the main route of ammonia assimilation in plants, and as a connection point between the organic and inorganic worlds. Even though GS activity is critical for producing high yields of crop plants, the autoregulation of substrate consumption of wheat GS remained unknown until now. Here we show kinetic evidence, that the chloroplast localized GS isoform (GS2 of wheat (Triticum aestivum L. cv. Jubilejnaja-50 takes place at the carbon-nitrogen metabolic branch point, where it is a mediator, and its enzymatic activity is regulated in a negatively cooperative allosteric manner. We have discovered that GS2 activity is described by a tetraphasic kinetic curve in response to increasing levels of glutamate supply. We constructed a model that explains the kinetic properties of glutamate consumption and this unique allosteric behavior. We also studied the subunit composition of both wheat leaf GS isoenzymes by a combination of two dimensional gel electrophoresis and protein blotting. Both leaf isozymes have homogeneous subunit composition. Glutamate is both a substrate, and an allosteric regulator of the biosynthetic reaction. We have concluded on the basis of our results and previous reports, that wheat GS2 is probably a homooctamer, and that it processes its substrate in a well-regulated, concentration dependent way, as a result of its negatively cooperative, allosteric activity. Thus, GS2 has a central role as a regulator between the nitrogen and the carbon cycles via maintaining glutamine-glutamate pool in the chloroplast on the level of substrates, in addition to its function in ammonia assimilation.

  1. The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin.

    Directory of Open Access Journals (Sweden)

    Amy J Gerc

    Full Text Available There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS assembly line (Alb4/5/6, tailoring enzymes (Alb2/3 and an export/resistance protein (Alb1, and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.

  2. Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP

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    Y. V. Preobrazhenskaya

    2013-01-01

    Full Text Available The transformation of the strain DH5αTM-T1R with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS, selD, from the E. coli BL21-Gold (DE3 strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′-O-(2,4,6-trinitrophenyladenosine-5′-triphosphate (TNP-ATP, was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with KDs different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.

  3. Astrocytes and Glutamate Homoeostasis in Alzheimer's Disease: A Decrease in Glutamine Synthetase, But Not in Glutamate Transporter-1, in the Prefrontal Cortex

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    Magdalena Kulijewicz-Nawrot

    2013-09-01

    Full Text Available Astrocytes control tissue equilibrium and hence define the homoeostasis and function of the CNS (central nervous system. Being principal homoeostatic cells, astroglia are fundamental for various forms of neuropathology, including AD (Alzheimer's disease. AD is a progressive neurodegenerative disorder characterized by the loss of cognitive functions due to specific lesions in mnesic-associated regions, including the mPFC (medial prefrontal cortex. Here, we analyzed the expression of GS (glutamine synthetase and GLT-1 (glutamate transporter-1 in astrocytes in the mPFC during the progression of AD in a triple-transgenic mouse model (3xTg-AD. GS is an astrocyte-specific enzyme, responsible for the intracellular conversion of glutamate into glutamine, whereas the removal of glutamate from the extracellular space is accomplished mainly by astroglia-specific GLT-1. We found a significant decrease in the numerical density (Nv, cells/mm3 of GS-positive astrocytes from early to middle ages (1–9 months; at the age of 1 month by 17%, 6 months by 27% and 9 months by 27% when compared with control animals in parallel with a reduced expression of GS (determined by Western blots, which started at the age of 6 months and was sustained up to 12 months of age. We did not, however, find any changes in the expression of GLT-1, which implies an intact glutamate uptake mechanism. Our results indicate that the decrease in GS expression may underlie a gradual decline in the vital astrocyte-dependent glutamate–glutamine conversion pathway, which in turn may compromise glutamate homoeostasis, leading towards failures in synaptic connectivity with deficient cognition and memory.

  4. Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

    Science.gov (United States)

    Seger, Mark; Gebril, Sayed; Tabilona, Jules; Peel, Amanda; Sengupta-Gopalan, Champa

    2015-01-01

    The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

  5. Liver-specific loss of long chain acyl-CoA synthetase-1 decreases triacylglycerol synthesis and beta-oxidation and alters phospholipid fatty acid composition.

    Science.gov (United States)

    Li, Lei O; Ellis, Jessica M; Paich, Heather A; Wang, Shuli; Gong, Nan; Altshuller, George; Thresher, Randy J; Koves, Timothy R; Watkins, Steven M; Muoio, Deborah M; Cline, Gary W; Shulman, Gerald I; Coleman, Rosalind A

    2009-10-09

    In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25-35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1(L)(-/-) liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [(14)C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [(14)C]oleate suggested that hepatic ACSL1 is important for mitochondrial beta-oxidation of long chain fatty acids. Because the Acsl1(L)(-/-) mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance.

  6. Liver-specific Loss of Long Chain Acyl-CoA Synthetase-1 Decreases Triacylglycerol Synthesis and β-Oxidation and Alters Phospholipid Fatty Acid Composition*

    Science.gov (United States)

    Li, Lei O.; Ellis, Jessica M.; Paich, Heather A.; Wang, Shuli; Gong, Nan; Altshuller, George; Thresher, Randy J.; Koves, Timothy R.; Watkins, Steven M.; Muoio, Deborah M.; Cline, Gary W.; Shulman, Gerald I.; Coleman, Rosalind A.

    2009-01-01

    In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25–35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1L−/− liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [14C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [14C]oleate suggested that hepatic ACSL1 is important for mitochondrial β-oxidation of long chain fatty acids. Because the Acsl1L−/− mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance. PMID:19648649

  7. One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia.

    Science.gov (United States)

    Sah, Shivjee; Aluri, Srinivas; Rex, Kervin; Varshney, Umesh

    2015-02-15

    In cells, N(10)-formyltetrahydrofolate (N(10)-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N(10)-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringens fhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (ΔfolD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N(10)-fTHF in the ΔfolD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N(10)-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The ΔfolD strain (harboring fhs on the chromosome) showed a high NADP(+)-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Two complementary enzymes for threonylation of tRNA in crenarchaeota: crystal structure of Aeropyrum pernix threonyl-tRNA synthetase lacking a cis-editing domain.

    Science.gov (United States)

    Shimizu, Satoru; Juan, Ella Czarina Magat; Sato, Yoshiteru; Miyashita, Yu-Ichiro; Hoque, Md Mominul; Suzuki, Kaoru; Sagara, Tsubasa; Tsunoda, Masaru; Sekiguchi, Takeshi; Dock-Bregeon, Anne-Catherine; Moras, Dino; Takénaka, Akio

    2009-11-27

    In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA(Thr) from different tRNAs in order to generate Thr-tRNA(Thr). In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA(Thr), one for catalysis and the other for trans-editing of misacylated Ser-tRNA(Thr). In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 A resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.

  9. The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

    Science.gov (United States)

    Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

    2012-01-01

    There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

  10. Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

    Science.gov (United States)

    Wakisaka, Shinji; Ohshima, Yoshifumi; Ogawa, Masahiro; Tochikura, Tatsurokuro; Tachiki, Takashi

    1998-01-01

    Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain. PMID:9687456

  11. Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

    DEFF Research Database (Denmark)

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...... on the promoter in front of the gcaD gene. Analysis of cDNA molecules prepared with gcaD-prs-ctc-specified mRNA as the template revealed an RNA transcript that encompassed all three cistrons....

  12. Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates

    Directory of Open Access Journals (Sweden)

    Rayevsky A. V.

    2016-02-01

    Full Text Available Aim. To investigate the structural bases for the amino acid selectivity of the Thermus thermophilus leucyl-tRNA synthetase (LeuRSTT aminoacylation site and to disclose the binding pattern of pre-transfer editing substrates. Methods. Eight amino acids proposed as semi-cognate substrates for aminoacylation and eight aminoacyl-adenylates (formed from AMP and eight amino acids were prepared in zwitterions form. The protein structure with a co-crystallized substrate in the aminoacylation site [PDBID: 1OBH] was taken from RCSB. Docking settings and evaluation of substrate efficiency were followed by twofold docking function analysis for each conformation with Gold CCDC. The molecular dynamics simulation was performed using Gromacs. The procedures of relaxation and binding study were separated in two different subsequent simulations for 50ns and 5ns. Results. The evaluation of substrate efficiency for 8 amino acids by twofold docking function analysis, based on score values,has shown that the ligands of LeuRSTT can be positioned in the following order: Leu>Nva>Hcy>Nle>Met>Cys>Ile >Val. MD simulation has revealed lower electrostatic interactions of isoleucine with the active site of the enzyme compared with those for norvaline and leucine. In the case of aminoacyl-adenylates no significant differences were found based on score values for both GoldScore and Asp functions. Molecular dynamics of leucyl-, isoleucyl- and norvalyl-adenylates showed that the most stable and conformationally favorable is leucine, then follow norvaline and isoleucine. It has been also found that the TYR43 of the active site covers carboxyl group of leucine and norvaline like a shield and deflected towards isoleucine, allowing water molecules to come closer. Conclusions. In this study we revealed some structural basis for screening unfavorable substrates by shape, size and flexibility of a radical. The results obtained for different amino acids by molecular docking and MD studies

  13. Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen.

    Science.gov (United States)

    Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

    2015-12-22

    Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the

  14. The multicenter study of a new assay for simultaneous detection of multiple anti-aminoacyl-tRNA synthetases in myositis and interstitial pneumonia.

    Directory of Open Access Journals (Sweden)

    Ran Nakashima

    Full Text Available OBJECTIVE: Autoantibodies to aminoacyl-tRNA synthetases (ARSs are useful in the diagnosis of idiopathic inflammatory myopathy (IIM with interstitial pneumonia (IP. We developed an enzyme-linked immunosorbent assay (ELISA system using a mixture of recombinant ARS antigens and tested its utility in a multicenter study. METHODS: We prepared six recombinant ARSs: GST-Jo-1, His-PL-12, His-EJ and GST-KS expressed in Escherichia coli, and His-PL-7 and His-OJ expressed in Hi-5 cells. After confirming their antigenic activity, with the exception of His-OJ, we developed our ELISA system in which the five recombinant ARSs (without His-OJ were mixed. Efficiency was confirmed using the sera from 526 Japanese patients with connective tissue disease (CTD (IIM n = 250, systemic lupus erythematosus n = 91, systemic sclerosis n = 70, rheumatoid arthritis n = 75, Sjögren's syndrome n = 27 and other diseases n = 13, 168 with idiopathic interstitial pneumonia (IIP and 30 healthy controls collected from eight institutes. IIPs were classified into two groups; idiopathic pulmonary fibrosis (IPF (n = 38 and non-IPF (n = 130. RESULTS were compared with those of RNA immunoprecipitation. RESULTS: Sensitivity and specificity of the ELISA were 97.1% and 99.8%, respectively when compared with the RNA immunoprecipitation assay. Anti-ARS antibodies were detected in 30.8% of IIM, 2.5% of non-myositis CTD, and 10.7% of IIP (5.3% of IPF and 12.3% of non-IPF. Anti-ARS-positive non-IPF patients were younger and more frequently treated with glucocorticoids and/or immunosuppressants than anti-ARS-negative patients. CONCLUSION: A newly established ELISA detected anti-ARS antibodies as efficiently as RNA immunoprecipitation. This system will enable easier and wider use in the detection of anti-ARS antibodies in patients with IIM and IIP.

  15. Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation.

    Science.gov (United States)

    Hu, Shufen; Xu, Weiyuan; Miao, Xiuhua; Gao, Yanping; Zhu, Liyan; Zhou, Youlang; Xiao, Ying; Xu, Guang-Yin

    2013-10-01

    The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS-H₂S signaling in sensitization of sodium channels in a previously validated rat model of IBS. Neonatal male rats (postnatal days 2-15) were exposed to a 3 hour period of daily maternal separation with temperature maintained at ~33 °C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely