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Sample records for acyl-coa synthetase acsl6

  1. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

    Directory of Open Access Journals (Sweden)

    Siliakus Melvin

    2010-04-01

    Full Text Available Abstract Background Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6. Results Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms. Conclusion The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.

  2. Chronic eosinophilic leukemia, NOS with t(5;12(q31;p13/ETV6-ACSL6 gene fusion: A novel variant of myeloid proliferative neoplasm with eosinophilia

    Directory of Open Access Journals (Sweden)

    Ruijun Jeanna Su

    2016-09-01

    Full Text Available The 2008 World Health Organization (WHO classification of tumors of hematopoietic and lymphoid tissues introduced a category for myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1. Many of these patients are responsive to tyrosine kinase inhibitor (TKI therapy. In this case report, we report a unique case of chronic eosinophilic leukemia with novel t(5;12(q23-31;p13/ETV6-ACSL6 gene fusion, in which patient was resistant to TKI therapy. This important finding is a novel addition to the above entity in WHO 2008 classification. The ACSL6 gene encodes a long-chain acyl-CoA synthetase, an enzyme that plays an essential role in lipid metabolism and ATP generation pathways in cells. The ETV6-ACSL6 rearrangement is present in diverse types of hematopoietic malignancies. As yet, it is not clear how ACSL6, a gene involved in fatty acid synthesis, contributes to clonal expansion of myeloid progenitor cells. Therefore, elucidating the contribution of ACSL6 to leukemogenesis may allow the development of novel treatment for those resistant to TKI therapy.

  3. Determination of hydrophobic coenzyme a esters and other lipids using a biosensor comprising a modified coenzyme a- and acyl-coa binding protein (acbp)

    DEFF Research Database (Denmark)

    2002-01-01

    , food and feed preparations, tissue extracts, acyl-CoA synthetase reaction media and various laboratory conditions using a modified Coenzyme A- and acyl-CoA binding protein (ACBP) is provided. Furthermore the invention relates to a construct comprising a peptide and a signal moiety for performing...

  4. 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...

  5. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

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    Wei-Ching Chen

    Full Text Available Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL

  6. Depletion of Arabidopsis ACYL-COA-BINDING PROTEIN3 Affects Fatty Acid Composition in the Phloem

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    Tai-Hua Hu

    2018-01-01

    Full Text Available Oxylipins are crucial components in plant wound responses that are mobilised via the plant vasculature. Previous studies have shown that the overexpression of an Arabidopsis acyl-CoA-binding protein, AtACBP3, led to an accumulation of oxylipin-containing galactolipids, and AtACBP3pro::BETA-GLUCURONIDASE (GUS was expressed in the phloem of transgenic Arabidopsis. To investigate the role of AtACBP3 in the phloem, reverse transcription-polymerase chain reaction and western blot analysis of phloem exudates from the acbp3 mutant and wild type revealed that the AtACBP3 protein, but not its mRNA, was detected in the phloem sap. Furthermore, micrografting demonstrated that AtACBP3 expressed from the 35S promoter was translocated from shoot to root. Subsequently, AtACBP3 was localised to the companion cells, sieve elements and the apoplastic space of phloem tissue by immunogold electron microscopy using anti-AtACBP3 antibodies. AtACBP3pro::GUS was induced locally in Arabidopsis leaves upon wounding, and the expression of wound-responsive jasmonic acid marker genes (JASMONATE ZIM-DOMAIN10, VEGETATIVE STORAGE PROTEIN2, and LIPOXYGENASE2 increased more significantly in both locally wounded and systemic leaves of the wild type in comparison to acbp3 and AtACBP3-RNAi. Oxylipin-related fatty acid (FA (C18:2-FA, C18:3-FA and methyl jasmonate content was observed to be lower in acbp3 and AtACBP3-RNAi than wild-type phloem exudates using gas chromatography-mass spectrometry. Experiments using recombinant AtACBP3 in isothermal titration calorimetry analysis showed that medium- and long-chain acyl-CoA esters bind (His6-AtACBP3 with KD values in the micromolar range. Taken together, these results suggest that AtACBP3 is likely to be a phloem-mobile protein that affects the FA pool and jasmonate content in the phloem, possibly by its binding to acyl-CoA esters.

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

    Science.gov (United States)

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

  8. 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...

  9. 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

  10. Physical studies of adenylosuccinate synthetase

    International Nuclear Information System (INIS)

    Bass, M.B.

    1987-01-01

    To determine the chemical mechanism of the reaction catalyzed by adenylosuccinate synthetase, positional isotope exchange studies were performed. Positional isotope exchange from the β-γ bridge to the β nonbridge position of [γ- 18 O]GTP was followed using 31 P NMR. The positional isotope exchange was found to occur in the presence of either IMP or IMP and succinate. The exchange did not occur in the presence of asparate. These results support a reaction mechanism which involves formation of a 6-phosphoryl-IMP intermediate with subsequent attack by aspartate to form adenylosuccinate as originally proposed by Lieberman in 1956. In order to resolve the NMR resonances for positional isotope exchange, it was necessary to find a chelator which would limit exchange broadening. trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid was found to be a suitable chelator at neutral and acidic pH. Studies of adenylosuccinate synthetase from Escherichia coli have been limited by the low concentrations of enzyme present in the cell and the difficulty in purifying the enzyme to homogeneity. Overproduction of the enzyme by cloning the purA gene into a runaway replication plasmid allowed the cells to produce a much higher concentration of enzyme. A new purification scheme is reported that takes advantage of the overproduced enzyme. Yields of 75 mg of homogeneous enzyme have been obtained from 76 g of E. coli cell paste

  11. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  12. Genetics Home Reference: phosphoribosylpyrophosphate synthetase superactivity

    Science.gov (United States)

    ... purines available. In people with the more severe form of PRS superactivity , PRPS1 gene mutations change single protein building blocks ( amino acids ) in the PRPP synthetase 1 enzyme, resulting in ...

  13. Disruption of the acyl-coa binding protein gene delays hepatic adaptation to metabolic changes at weaning

    DEFF Research Database (Denmark)

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek

    2011-01-01

    The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an intracellular protein that binds C14-C22 acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however...

  14. Glutamine Synthetase: Localization Dictates Outcome

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    Alessandra Castegna

    2018-02-01

    Full Text Available Glutamine synthetase (GS is the adenosine triphosphate (ATP-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event. In vitro evidence, confirmed in vivo in some cases, suggests that reduced GS activity in cancer cells associates with a more invasive and aggressive phenotype. However, GS is known to be highly expressed in cells of the tumor microenvironment, such as fibroblasts, adipocytes and immune cells, and their ability to synthesize glutamine is responsible for the acquisition of protumoral phenotypes. This has opened a new window into the complex scenario of the tumor microenvironment, in which the balance of glutamine consumption versus glutamine synthesis influences cellular function. Since GS expression responds to glutamine starvation, a lower glutamine synthesizing power due to the absence of GS in cancer cells might apply a metabolic pressure on stromal cells. This event might push stroma towards a GS-high/protumoral phenotype. When referred to stromal cells, GS expression might acquire a ‘bad’ significance to the point that GS inhibition might be considered a conceivable strategy against cancer metastasis.

  15. The glycyl-tRNA synthetase of Chlamydia trachomatis.

    Science.gov (United States)

    Wagar, E A; Giese, M J; Yasin, B; Pang, M

    1995-01-01

    Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids. A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames. We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS). This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene. PMID:7665503

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

    African Journals Online (AJOL)

    Khaled

    2012-07-10

    Jul 10, 2012 ... affected by growth medium, carbon source, nitrogen source and sodium chloride. LB supplemented with 7% glycerol ... Abbreviations: GS, Glutamine synthetase; MSM, minimal salt medium; NB, nutrient broth medium; NF, ... glutamate and ammonia, which in turn, cells are supplied with ammonia, and their ...

  17. Phosphinothricin-tripeptide synthetases from Streptomyces viridochromogenes.

    Science.gov (United States)

    Grammel, N; Schwartz, D; Wohlleben, W; Keller, U

    1998-02-10

    Phosphinothricyl-alanyl-alanine (Pt tripeptide (Ptt), bialaphos) is a metabolite produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. It contains the unique phosphinoamino acid phosphinothricin (Pt), which after cleavage from Ptt is active as an inhibitor of glutamine synthetase. We have isolated three enzymes that assemble the building block of the Ptt peptide backbone in a nonribosomal mechanism. The first enzyme, named Ptt-synthetase I (PTTS I), activates N-acetyldemethylphosphinothricin (AcDMPt) as adenylate and thioester. Pt is not activated. PTTS I can also activate N-acetylphosphinothricin (AcPt) or N-acetylglutamate as structural analogues of AcDMPT. Native PTTS I has an estimated size of 62 kDa whereas the denatured form displays a size of 76 kDa. Immunoblot analysis and determination of its N-terminal protein sequence revealed that PTTS I is identical with the gene product of phsA. The phsA gene was previously identified near the Pt-resistance gene pat in the Ptt biosynthesis gene cluster in S. viridochromogenes. Besides PTTS I, two alanine-activating enzymes (PTTS II/III) were partially purified from S. viridochromogenes with estimated native sizes of ca. 120 kDa (enzyme 1) and ca. 140 kDa (enzyme 2). Both enzymes bind alanine as a thioester via the corresponding adenylate. Level of PTTS II/III and product formation were correlated with each other in several different strains of S. viridochromogenes. These results indicate that Ptt is synthesized by three peptide synthetases, each activating one single amino acid. The data also confirm previous genetic data, which suggest that AcDMPt-Ala-Ala is the precursor of Ptt.

  18. Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.

    OpenAIRE

    Johnson, D L; Yang, D C

    1981-01-01

    The particulate aminoacyl-tRNA synthetases of rat liver were copurified about 1000-fold with more than 20% yields for individual synthetase activities. Measurements of aminoacylation activities showed that lysyl-, arginyl-, leucyl-, isoleucyl-, and methionyl-tRNA synthetases in the purified complex cosedimented at 18 S. The molecular weight of the synthetase complex is about one million, as estimated by gel filtration. The stoichiometry of the synthetase in the complex was determined by activ...

  19. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    Science.gov (United States)

    ... hyperammonemia, type I Genetics Education Materials for School Success (GEMSS) Orphanet: Carbamoyl-phosphate synthetase 1 deficiency Patient ... for Links Data Files & API Site Map Subscribe Customer Support USA.gov Copyright Privacy Accessibility FOIA Viewers & ...

  20. New perspectives on glutamine synthetase in grasses.

    Science.gov (United States)

    Swarbreck, Stéphanie M; Defoin-Platel, M; Hindle, M; Saqi, M; Habash, Dimah Z

    2011-02-01

    Members of the glutamine synthetase (GS) gene family have now been characterized in many crop species such as wheat, rice, and maize. Studies have shown that cytosolic GS isoforms are involved in nitrogen remobilization during leaf senescence and emphasized a role in seed production particularly in small grain crop species. Data from the sequencing of genomes for model crops and expressed sequence tag (EST) libraries from non-model species have strengthened the idea that the cytosolic GS genes are organized in three functionally and phylogenetically conserved subfamilies. Using a bioinformatic approach, the considerable publicly available information on high throughput gene expression was mined to search for genes having patterns of expression similar to GS. Interesting new hypotheses have emerged from searching for co-expressed genes across multiple unfiltered experimental data sets in rice. This approach should inform new experimental designs and studies to explore the regulation of the GS gene family further. It is expected that understanding the regulation of GS under varied climatic conditions will emerge as an important new area considering the results from recent studies that have shown nitrogen assimilation to be critical to plant acclimation to high CO(2) concentrations.

  1. Gleaning unexpected fruits from hard-won synthetases: probing principles of permissivity in non-canonical amino acid-tRNA synthetases.

    Science.gov (United States)

    Cooley, Richard B; Karplus, P Andrew; Mehl, Ryan A

    2014-08-18

    The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins is an important tool for understanding biological function. Traditionally, each new ncAA targeted for incorporation requires a resource-consuming process of generating new ncAA aminoacyl tRNA synthetase/tRNACUA pairs. However, the discovery that some tRNA synthetases are "permissive", in that they can incorporate multiple ncAAs, means that it is no longer always necessary to develop a new synthetase for each newly desired ncAA. Developing a better understanding of what factors make ncAA synthetases more permissive would increase the utility of this new approach. Here, we characterized two synthetases selected for the same ncAA that have markedly different "permissivity profiles." Remarkably, the more permissive synthetase incorporated an ncAA for which we had not been able to generate a synthetase through de novo selection methods. Crystal structures revealed that the two synthetases recognize their parent ncAA through a conserved core of interactions, with the more permissive synthetase displaying a greater degree of flexibility in its interaction geometries. We also observed that intraprotein interactions not directly involved in ncAA binding can play a crucial role in synthetase permissivity and suggest that optimization of such interactions might provide an avenue to engineering synthetases with enhanced permissivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-12-13

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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Regions involved in fengycin synthetases enzyme complex formation

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Cheng

    2017-12-01

    Full Text Available Background: Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions. Methods: Deletions were generated in the fengycin synthetases. The interaction of these mutant proteins with their partner enzymes in the complex was analyzed in vitro by a glutathione S-transferase (GST or nickel pulldown assay. Results: The communication-mediating donor (COM-D domains of the fengycin synthetases, when fused to GST, specifically pulled down their downstream partner enzymes in the GST-pulldown assays. The communication-mediating acceptor (COM-A domains were required for binding between two partner enzymes, although the domains alone did not confer specificity of the binding to their upstream partner enzymes. This study found that the COM-A domain, the condensation domain, and a portion of the adenylation domain in fengycin synthetase B (FenB were required for specific binding to fengycin synthetase A (FenA. Conclusion: The interaction between the COM-D and COM-A domains in two partner enzymes is critical for nonribosomal peptide synthesis. The COM-A domain alone is insufficient for interacting with its upstream partner enzyme in the enzyme complex with specificity; a region that contains COM-A, condensation, and a portion of adenylation domains in the downstream partner enzyme is required. Keywords: communication-mediating donor and acceptor domain, fengycin synthetase, protein-protein interaction

  8. 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...

  9. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    2017-01-19

    Jan 19, 2017 ... 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 impor- tant antibiotics and siderophores. Each of the multiple modules of an ...

  10. Heterogeneous distribution of glutamine synthetase during rat liver development

    NARCIS (Netherlands)

    Gaasbeek Janzen, J. W.; Gebhardt, R.; ten Voorde, G. H.; Lamers, W. H.; Charles, R.; Moorman, A. F.

    1987-01-01

    Two days before birth, immunohistochemical detection of glutamine synthetase already reveals a heterogeneous distribution pattern related to the vascular architecture of the liver. Only a small number of hepatocytes in the vicinity of the efferent venules show relatively high staining intensity.

  11. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are themajor multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically importantantibiotics and siderophores. Each of the multiple modules of an NRPS activates a ...

  12. 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.

  13. Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Vermeulen, Jacqueline L. M.; Labruyère, Wilhelmina T.; de Waart, D. Rudi; van der Hel, W. Saskia; Ruijter, Jan M.; Uylings, Harry B. M.; Lamers, Wouter H.

    2010-01-01

    Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/f)l mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in

  14. 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 ...

  15. 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.

  16. Genomic, Biochemical, and Modeling Analyses of Asparagine Synthetases from Wheat

    Directory of Open Access Journals (Sweden)

    Hongwei Xu

    2018-01-01

    Full Text Available Asparagine synthetase activity in cereals has become an important issue with the discovery that free asparagine concentration determines the potential for formation of acrylamide, a probably carcinogenic processing contaminant, in baked cereal products. Asparagine synthetase catalyses the ATP-dependent transfer of the amino group of glutamine to a molecule of aspartate to generate glutamate and asparagine. Here, asparagine synthetase-encoding polymerase chain reaction (PCR products were amplified from wheat (Triticum aestivum cv. Spark cDNA. The encoded proteins were assigned the names TaASN1, TaASN2, and TaASN3 on the basis of comparisons with other wheat and cereal asparagine synthetases. Although very similar to each other they differed slightly in size, with molecular masses of 65.49, 65.06, and 66.24 kDa, respectively. Chromosomal positions and scaffold references were established for TaASN1, TaASN2, and TaASN3, and a fourth, more recently identified gene, TaASN4. TaASN1, TaASN2, and TaASN4 were all found to be single copy genes, located on chromosomes 5, 3, and 4, respectively, of each genome (A, B, and D, although variety Chinese Spring lacked a TaASN2 gene in the B genome. Two copies of TaASN3 were found on chromosome 1 of each genome, and these were given the names TaASN3.1 and TaASN3.2. The TaASN1, TaASN2, and TaASN3 PCR products were heterologously expressed in Escherichia coli (TaASN4 was not investigated in this part of the study. Western blot analysis identified two monoclonal antibodies that recognized the three proteins, but did not distinguish between them, despite being raised to epitopes SKKPRMIEVAAP and GGSNKPGVMNTV in the variable C-terminal regions of the proteins. The heterologously expressed TaASN1 and TaASN2 proteins were found to be active asparagine synthetases, producing asparagine and glutamate from glutamine and aspartate. The asparagine synthetase reaction was modeled using SNOOPY® software and information from

  17. A Rare Cause of Neonatal Hemolytic Anemia: Glutathione Synthetase Deficiency.

    Science.gov (United States)

    Soylu Ustkoyuncu, Pembe; Mutlu, Fatma Türkan; Kiraz, Aslihan; Tag Balkis, Zuhal; Yel, Sibel

    2018-01-01

    Isolated hemolysis or hemolytic anemia and 5-oxoprolinuria are 2 distinct medical conditions in the clinical spectrum associated with glutathione synthetase deficiency. A 1-day-old female baby presented with anemia and respiratory distress. Her hemoglobin level was 9.5 g/dL and the total serum bilirubin level was 5.6 mg/dL. Metabolic acidosis was detected in her blood gas analysis. Metabolic acidosis recurred despite treatment and further investigation was required. Her 5-oxoproline level was 3815 mmol/mol creatinine in urine organic acid analysis, and a homozygous mutation [p.R125H (c.374G>A)] was found in the glutathione synthetase gene. GSD has been observed in very few patients and is rarely considered in the differential diagnosis of hemolytic anemia in newborns.

  18. Glutamine synthetase gene evolution: A good molecular clock

    International Nuclear Information System (INIS)

    Pesole, G.; Lanvave, C.; Saccone, C.; Bozzetti, M.P.; Preparata, G.

    1991-01-01

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves

  19. 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.

  20. 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.

  1. The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

    Science.gov (United States)

    Bieganowski, Pawel; Brenner, Charles

    2003-08-29

    Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

  2. 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

  3. Phosphinothricin tripeptide synthetases in Streptomyces viridochromogenes Tü494.

    Science.gov (United States)

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

    2005-11-01

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

  4. 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....

  5. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

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

    Science.gov (United States)

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

    2011-09-06

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

  7. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... of these enzymes for correct genetic code expression as well as early structural data and related enzymology will be reviewed. Despite structural diversity, all synthetases follow a two-step mechanism for tRNA aminoacylation. Specificity, however, is not absolute since synthetases were shown to catalyze ...

  8. [The anti-synthetase syndrome: muscle disease and multisystem disorder at the same time

    NARCIS (Netherlands)

    Hengstman, G.J.D.; Venrooij, W.J.W. van; Hoogen, F.H.J. van den; Engelen, B.G.M. van

    2003-01-01

    In three women, aged 60, 45 and 38 years, who presented with exertional dyspnoea (due to lung fibrosis) and Raynaud's phenomenon, dermatomyopathy and Raynaud's phenomenon, and symmetrical arthralgia and myalgia, respectively, the anti-synthetase syndrome was diagnosed. The anti-synthetase syndrome

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

    Science.gov (United States)

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

    2008-04-08

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

  10. Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

    Science.gov (United States)

    Shiba, K; Motegi, H; Yoshida, M; Noda, T

    1998-11-15

    We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

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

    Science.gov (United States)

    Pyne, C; Bognar, A L

    1992-03-01

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

  12. 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.

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

    International Nuclear Information System (INIS)

    Ikeda, Masamichi; Iwai, Kazuo

    1976-01-01

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg 2+ and K + as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

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

    International Nuclear Information System (INIS)

    Bandaralage, Sahan P.S.; Farnaghi, Soheil; Dulhunty, Joel M.; Kothari, Alka

    2016-01-01

    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.)

  15. 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.)

  16. 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

  17. Allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase in Clostridium: another conserved generic characteristic.

    Science.gov (United States)

    Jensen, R A; Twarog, R

    1972-09-01

    Enzymological studies were done to characterize the allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase in three species of Clostridium. Allosteric control was identified as feedback inhibition by phenylalanine and was qualitatively similar for the DAHP synthetases of C. butyricum, C. acetobutylicum, and C. tetanomorphum. Quantitative differences in the enzymology and kinetics of allosteric control distinguished C. tetanomorphum from C. butyricum and C. acetobutylicum. Crude extracts contained apparent proteolytic activity which could be fractionated from DAHP synthetase. The proteolytic activity was more labile than DAHP synthetase in extracts and was progressively inactivated by serial freeze-thaw treatments. Protease activity was at least partially inhibited by phenylmethylsulfonyl-fluoride. The method of comparative allostery of DAHP synthetase distinguishes the genera Bacillus and Clostridium, each having a strongly conserved pattern of regulation for DAHP synthetase. The data reinforce previous conclusions that allosteric control patterns governing the activity of DAHP synthetase are stable, reliable generic characteristics.

  18. 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.

  19. 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)

  20. 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.

  1. 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...

  2. 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

  3. REPRESSION BY ADENINE OF THE FORMYLTETRAHYDROFOLATE SYNTHETASE IN AN ANTIFOLIC-RESISTANT MUTANT OF STREPTOCOCCUS FAECALIS.

    Science.gov (United States)

    ALBRECHT, A M; HUTCHISON, D J

    1964-04-01

    Albrecht, Alberta M. (Sloan-Kettering Institute for Cancer Research, New York, N.Y.), and Dorris J. Hutchison. Repression by adenine of the formyltetrahydrofolate synthetase in an antifolic-resistant mutant of Streptococcus faecalis. J. Bacteriol. 87:792-798. 1964.-In an amethopterin-resistant mutant of Streptococcus faecalis ATCC 8043 under cultivation conditions requiring purine synthesis de novo, both the dihydrofolate reductase and the formyltetrahydrofolate synthetase were formed as constant fractions of the total protein synthesized during the exponential phase of growth. When excess adenine was added to the medium, the rate of formation of the synthetase was markedly decreased, i.e., repressed. Under these latter conditions, the synthesis of the reductase proceeded at a rate equal to that observed in the absence of adenine. The repressibility of the synthetase by adenine was demonstrated also by the decrease in rate of synthetase formation upon the addition of adenine to a culture actively synthesizing this enzyme. Guanine and hypoxanthine, like adenine, also repressed the synthetase; exogenous xanthine was less effective. Neither of the pyrimidines, thymine and uracil, at approximately 1 mug/ml, interfered with synthesis of the two enzymes.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-30

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

  5. Characterization of FdmV as an Amide Synthetase for Fredericamycin A Biosynthesis in Streptomyces griseus ATCC 43944*

    OpenAIRE

    Chen, Yihua; Wendt-Pienkowski, Evelyn; Ju, Jianhua; Lin, Shuangjun; Rajski, Scott R.; Shen, Ben

    2010-01-01

    Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the...

  6. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  7. 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.

  8. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    International Nuclear Information System (INIS)

    Das, S.; Gillin, F.D.

    1987-01-01

    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 3 H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei

  9. 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.

  10. Essential nontranslational functions of tRNA synthetases.

    Science.gov (United States)

    Guo, Min; Schimmel, Paul

    2013-03-01

    Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. Although these new functions were thought to be 'moonlighting activities', many are as critical for cellular homeostasis as their activity in translation. New roles have been associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect pathways for cardiovascular development and the immune response and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. New architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. Although a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid-binding site for another purpose.

  11. Essential Non-Translational Functions of tRNA Synthetases

    Science.gov (United States)

    Guo, Min; Schimmel, Paul

    2013-01-01

    Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. While these new functions were thought to be ‘moonlighting activities’, many are as critical for cellular homeostasis as the activity in translation. New roles have been associated with cytoplasmic forms as well as with nuclear and secreted extracellular forms that impact pathways for cardiovascular development, the immune response, and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. Novel architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. While a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid binding site for another purpose. PMID:23416400

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    A Theron

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

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

    Science.gov (United States)

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

    2017-01-01

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

  16. Valyl-tRNA synthetase gene of Escherichia coli K12: Molecular genetic characterization and homology within a family of aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Heck, J.D. III.

    1988-01-01

    This work reports the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene encoding valyl-tRNA synthetase. The valS gene was subcloned from plasmid pLC26-22 by genetic complementation of a valS ts strain. The DNA region encoding the valS structural gene was determined by in vitro coupled transcription-translation assays. Cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase specific activity twelve-fold. DNA sequences flanking the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both [α- 32 P]labeled and [γ- 32 P]end-labeled in vitro transcription assays. The DNA sequence of the valS gene of Escherichia coli has been determined. Significant similarity at the primary sequence level was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. An extended open reading frame (ORF) encoded on the DNA strand opposite the valS structural gene is described

  17. Differential expression of argininosuccinate synthetase in serous and non‐serous ovarian carcinomas

    Science.gov (United States)

    Cheon, Dong‐Joo; Walts, Ann E; Beach, Jessica A; Lester, Jenny; Bomalaski, John S; Walsh, Christine S; Ruprecht Wiedemeyer, W; Karlan, Beth Y

    2014-01-01

    Abstract The current standard of care for epithelial ovarian cancer does not discriminate between different histologic subtypes (serous, clear cell, endometrioid and mucinous) despite the knowledge that ovarian carcinoma subtypes do not respond uniformly to conventional platinum/taxane‐based chemotherapy. Exploiting addictions and vulnerabilities in cancers with distinguishable molecular features presents an opportunity to develop individualized therapies that may be more effective than the current ‘one size fits all' approach. One such opportunity is arginine depletion therapy with pegylated arginine deiminase, which has shown promise in several cancer types that exhibit low levels of argininosuccinate synthetase including hepatocellular and prostate carcinoma and melanoma. Based on the high levels of argininosuccinate synthetase previously observed in ovarian cancers, these tumours have been considered unlikely candidates for arginine depletion therapy. However, argininosuccinate synthetase levels have not been evaluated in the individual histologic subtypes of ovarian carcinoma. The current study is the first to examine the expression of argininosuccinate synthetase at the mRNA and protein levels in large cohorts of primary and recurrent ovarian carcinomas and ovarian cancer cell lines. We show that the normal fallopian tube fimbria and the majority of primary high‐grade and low‐grade serous ovarian carcinomas express high levels of argininosuccinate synthetase, which tend to further increase in recurrent tumours. In contrast to the serous subtype, non‐serous ovarian carcinoma subtypes (clear cell, endometrioid and mucinous) frequently lack detectable argininosuccinate synthetase expression. The in vitro sensitivity of ovarian cancer cell lines to arginine depletion with pegylated arginine deiminase was inversely correlated with argininosuccinate synthetase expression. Our data suggest that the majority of serous ovarian carcinomas are not susceptible

  18. Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (Opsanus beta).

    Science.gov (United States)

    Kong, H; Kahatapitiya, N; Kingsley, K; Salo, W L; Anderson, P M; Wang, Y S; Walsh, P J

    2000-01-01

    Gulf toadfish (Opsanus &bgr;) rapidly switch to excretion of urea as their main nitrogenous waste product under several laboratory conditions, including confinement to small volumes of water. Prior evidence suggested that the activities of two key enzymes of urea synthesis exhibited potentially different modes of upregulation during this switch, with carbamoyl phosphate synthethase III (CPSase III) activated allosterically by N-acetylglutamate, and glutamine synthetase (GSase) activated by increases in the concentration of protein. The present study was undertaken to examine additional aspects of the regulation of these enzymes. The sequence for O. beta CPSase III cDNA was obtained, and it was found to be similar to that of other piscine CPSases. The sequence also allowed us to develop riboprobes for CPSase III mRNA analysis using ribonuclease protection assays (RPAs). CPSase III mRNA was expressed in liver, muscle, kidney and intestine, in agreement with prior enzymatic measurements. Levels of CPSase III mRNA increased five- to tenfold (relative to beta-actin mRNA) in liver (but not muscle) following 48 h of confinement stress. Measured by western analysis using an antibody to chicken GSase, confined O. beta GSase protein concentrations increased eightfold over control levels, in agreement with prior and present measurements of increases in GSase activity. Furthermore, RPAs of GSase mRNA levels demonstrated an increase of fivefold during confinement.

  19. 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

  20. 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.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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

  2. Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

    Science.gov (United States)

    Mathur, M; Satpathy, M; Sachar, R C

    1992-11-17

    Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments

  3. Identification of autoantibodies to tyrosil-tRNA synthetase in heart disfunctions

    Directory of Open Access Journals (Sweden)

    Ryabenko D. V.

    2010-09-01

    Full Text Available Aim. To investigate the levels of specific autoantibodies against tyrosyl-tRNA synthetase and its individual modules in the blood serum of people with heart failure caused by dilated cardiomyopathy, myocarditis and ischemic heart disease compared with healthy donors. Methods. Recombinant proteins were obtained using bacterial strains transformed with appropriate plasmid vectors and were purified by chromatography on Ni-NTA-agarose. The levels of specific autoantibodies were investigated by ELISA. Results. The increased levels of autoantibodies specific to tyrosyl-tRNA synthetase, its N-terminal catalytic module and non-catalytic C-module, were found in the blood serum of patients, compared with healthy donors. Conclusions. The results obtained demonstrate the possible role of tyrosyl-tRNA synthetase in adaptive changes of the myocardium in response to stress factors.

  4. Inhibition of Dihydropteroate Synthetase from Escherichia coli by Sulfones and Sulfonamides

    Science.gov (United States)

    McCullough, Jerry L.; Maren, Thomas H.

    1973-01-01

    The inhibitory action of various diphenylsulfones and sulfonamides on dihydropteroate synthetase partially purified from Escherichia coli was examined. 4,4′-Diaminodiphenylsulfone (DDS; I50 = 2 × 10−5 M) and the monosubstituted derivatives 4-amino-4′-formamidodiphenylsulfone (I50 = 5.8 × 10−5 M) and 4-amino-4′-acetamidodiphenylsulfone (I50 = 5.2 × 10−5 M) were effective inhibitors of dihydropteroate synthetase activity. Disubstitution of the arylamine groups of DDS (4,4′-diformamidodiphenylsulfone and 4,4′-diacetamidodiphenylsulfone) resulted in complete loss of inhibitory activity. Both DDS (KI = 5.9 × 10−6 M) and sulfadiazine (KI = 2.5 × 10−6 M) were found to be competitive inhibitors of dihydropteroate synthetase. These findings are discussed in regard to the Bell and Roblin theory of structure-activity relationships for p-aminobenzoic acid antagonists. PMID:4597736

  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. Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Charles, R.; Lamers, W. H.

    1990-01-01

    A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary-treated rats was carried out, using radioactively-labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line

  7. 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...

  8. 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

    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 produc...

  9. The influence of prenatal X-irradiation on the activity of SRNA-aminoacyl synthetases in the developing rabbit brain

    International Nuclear Information System (INIS)

    Wender, M.; Zgorzalewicz, B.

    1976-01-01

    The activities of sRNA-aminoacyl synthetases were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single x-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. The results indicate clearly that prenatal exposure to ionizing radiation also affects the steps of protein biosynthesis which depend on the activity of sRNA-aminoacyl synthetases. (author)

  10. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    -utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5......-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate...

  11. 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 translatio...... of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions....

  12. 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...

  13. 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

  14. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

    2016-01-01

    Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

  15. 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 se...

  16. 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

  17. Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase.

    NARCIS (Netherlands)

    Guan, C.; Ribeiro, A.; Akkermans, A.D.L.; Jing, Y.; Kammen, van A.; Bisseling, T.; Pawlowski, K.

    1996-01-01

    Two nodule cDNA clones representing genes involved in Alnus glutinosa nitrogen metabolism were analysed. ag11 encoded glutamine synthetase (GS), the enzyme responsible for ammonium assimilation, while ag118 encoded acetylornithine transaminase (AOTA), an enzyme involved in the biosynthesis of

  18. 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......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 purified by ammonium sulfate precipitation and DEAE-cellulose chromatography and separated into four fractions. In the absence of an added carbohydrate primer two of the four fractions catalized the synthesis of a methanol-precipitable agr-glucan when high concentrations of sodium citrate and bovine serum...... albumim were added. The rate of agr-glucan synthesis by the unprimed reaction was higher than for the primed reaction. The four enzyme fractions were active with ADP-Glc, but not with UDP-Glc, both in the primed and in the unprimed reaction....

  19. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  20. 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

  1. Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum

    NARCIS (Netherlands)

    Di Lorenzo, M.; Stork, M.; Naka, H.; Tolmasky, M.E.; Crosa, J.H.

    2008-01-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa

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

    NARCIS (Netherlands)

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

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of

  3. Glutamine Synthetase in Muscle Is Required for Glutamine Production during Fasting and Extrahepatic Ammonia Detoxification

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Köhler, S. Eleonore; Vermeulen, Jacqueline L. M.; de Waart, D. Rudi; de Theije, Chiel; ten Have, Gabrie A. M.; van Eijk, Hans M. H.; Kunne, Cindy; Labruyere, Wilhelmina T.; Houten, Sander M.; Sokolovic, Milka; Ruijter, Jan M.; Deutz, Nicolaas E. P.; Lamers, Wouter H.

    2010-01-01

    The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with

  4. Expression pattern of glutamine synthetase marks transition from collecting into conducting hepatic veins

    NARCIS (Netherlands)

    Lamers, W. H.; Vermeulen, J. L.; Hakvoort, T. B.; Moorman, A. F.

    1999-01-01

    The expression of glutamine synthetase (GS) is confined to a rim of hepatocytes surrounding the efferent hepatic veins in all mammalian species investigated. In rat liver, a two- to three-cell thick layer of GS-positive (GS(+)) hepatocytes uniformly surrounds the two to four terminal branching

  5. 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

  6. Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

    Directory of Open Access Journals (Sweden)

    Zheng-xun JIN

    2007-09-01

    Full Text Available Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

  7. 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...

  8. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne U.; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine...

  9. Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

    Science.gov (United States)

    Aboelnga, Mohamed M.

    Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre?transfer editing and post?transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyl-tRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates

  10. Equilibria and partitioning of complexes in the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.

    1987-01-01

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a reaction in which the [enzyme-ATP-methionine] complex reacts to form an intermediate [enzyme-AdoMet-PPPi] complex: hydrolysis of PPPi yields an [enzyme-AdoMet-PPi-Pi] complex from which AdoMet is the last product to dissociate. Analysis of reaction mixtures which were quenched with acid during turnover of E. coli AdoMet synthetase with saturating substrates containing [α - 32 P]ATP showed that PPPi is present in an amount corresponding to 45% of the total enzyme active sites, reflecting the portion of enzyme present in an [enzyme-AdoMet-PPPi] complex. Similar experiments in which excess pyrophosphatase was included (to hydrolyze PPi as it was released from AdoMet synthetase), showed that enzyme-bound PPi is present in an amount corresponding to 22% of the total AdoMet synthetase. The enzyme not present in complexes with PPPi or PPi is probably distributed between the [enzyme-ATP-methionine] and the [enzyme-AdoMet] complexes. AdoMet synthetase forms enzyme-bound 32 PPPi from added 32 PPi and Pi; the equilibrium constant [enzyme-AdoMet-PPi-Pi]/[enzyme-AdoMet-PPPi] is 2.0, greatly displaced from the equilibrium for hydrolysis of free PPPi. Since the ratio of enzyme-bound PPi to PPPi is 0.5 during the steady state, the PPPi hydrolysis step is not at equilibrium during turnover. Formation of [ 32 P]ATP from the [enzyme-AdoMet- 32 PPPi] complex was not detected

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

    Energy Technology Data Exchange (ETDEWEB)

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara (SSRL); (Maryland)

    2012-08-31

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

  12. 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

    of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined......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...

  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. 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 studies indicating an alternative mechanism via the cytochrome cytochrome bc1 complex impacting on the homeostasis of ATP synthesis [39]. The inhibition of glutamine synthetase may also impact the ATP homeostasis as the resultant accumulation of α...

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

    Science.gov (United States)

    Anderson, J Christopher [San Francisco, CA; Wu, Ning [Brookline, MA; Santoro, Stephen [Cambridge, MA; Schultz, Peter G [La Jolla, CA

    2009-08-18

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

  16. 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.

  17. Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

    Science.gov (United States)

    Lee, Eun-Young; Lee, Hyun-Cheol; Kim, Hyun-Kwan; Jang, Song Yee; Park, Seong-Jun; Kim, Yong-Hoon; Kim, Jong Hwan; Hwang, Jungwon; Kim, Jae-Hoon; Kim, Tae-Hwan; Arif, Abul; Kim, Seon-Young; Choi, Young-Ki; Lee, Cheolju; Lee, Chul-Ho; Jung, Jae U; Fox, Paul L; Kim, Sunghoon; Lee, Jong-Soo; Kim, Myung Hee

    2016-01-01

    The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/−) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection. PMID:27595231

  18. Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

    Science.gov (United States)

    Sharma, Arvind; Sharma, Amit

    2015-02-01

    The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

  19. 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.

  20. 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.

  1. Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

    International Nuclear Information System (INIS)

    Chu, Wenchy; Horowitz, J.

    1991-01-01

    Interactions of 5-fluorouracil-substituted Escherichia coli tRNA Val with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K M and V max values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA Val . Binding of VRS to (FUra)tRNA Val induces structural perturbations that are reflected in selective changes in the 19 F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA Val along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA Val , suggesting conformational changes in this part of the molecule. No 19 F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA Val that has been proposed as a common intermediate in the aminoacylation reaction

  2. 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 promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::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.

  3. Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus).

    Science.gov (United States)

    Dalessandro, G; Northcote, D H

    1981-01-01

    Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-(14)C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-(14)C]xylose released all the radioactivity as xylose. β-1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4-β xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[(14)C]xylose and tri-O-methyl-[(14)C]xylose, suggesting a 1→4-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent Km value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg(2+) and Mn(2+) ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.

  4. 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.

  5. Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium

    DEFF Research Database (Denmark)

    Hansen, Jørgen T.; Sørensen, Jens L.; Giese, Henriette

    2012-01-01

    for future polyketide synthases (PKSs) and nonribosomal peptides synthetases (NRPSs) nomenclature assignment and classification. Sequence similarities of the adenylation and ketosynthase domain sequences were used to group the identified NRPS and PKS genes. We present the current state of knowledge of PKS......Fusarium species produce a plethora of bioactive polyketides and nonribosomal peptides that give rise to health problems in animals and may have drug development potential. Using the genome sequences for Fusarium graminearum, F. oxysporum, F. solani and F. verticillioides we developed a framework...

  6. Evolution of the 2'-5'-Oligoadenylate Synthetase family in eukaryotes and bacteria

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Poulsen, Jesper Buchhave; Reitamm, Tonu

    2009-01-01

    system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins......The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon...

  7. 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...... represented a primary stimulus in all subjects. First, basal 2',5'A activity increased severalfold in response to yellow fever vaccination. In IDDM subjects, this increase was significantly lower (P = .025). Second, the 2',5'A activity increased proportionately to the higher basal 2',5'A activity in IDDM...

  8. 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.

  9. 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...

  10. 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

    -dependent methyltransferase 144, GTP cyclohydrolase 398, DNA topoisomerase VI subunit A 209, DNA topoisomerase VI subunit B 192, Type A Flavoprotein 911, NAD(P)H:rubredoxin oxidoreductase (Fatty acid metabolism) 120, NAD(P)H:rubredoxin oxidoreductase 120, cofactor-independent phosphoglycerate mutase 909, bis(5'-adenosyl......, transcriptional regulator 364, glutamine synthetase 120, N6-adenine-specific DNA methylase 194, ArsR family transcriptional regulator 113, 5'-methylthioadenosine phosphorylase II 280, DNA repair and recombination protein RadA 323, 30S ribosomal protein S6e 106, pyruvate ferredoxin oxidoreductase subunit beta 282...

  11. 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.

  12. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  13. 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...

  14. δ-(L-α-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    Lende, Ted R. van der; Kamp, Mart van de; Berg, Marco van den; Sjollema, Klaas; Bovenberg, Roel A.L.; Veenhuis, Marten; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-α-aminoadipate, L-cysteine, and L-valine into

  15. delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    van der Lende, T.R.; de Kamp, M.; den Berg, M.van; Sjollema, K.; Bovenberg, R.A.L.; Veenhuis, M; Konings, W.N; Driessen, A.J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteme, and

  16. 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...

  17. 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...

  18. 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.

  19. 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...

  20. 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

    Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology......-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine....

  1. β-Keto and β-hydroxyphosphonate analogs of biotin-5’-AMP are inhibitors of holocarboxylase synthetase

    OpenAIRE

    Sittiwong, Wantanee; Cordonier, Elizabeth L.; Zempleni, Janos; Dussault, Patrick H.

    2014-01-01

    Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5′-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 μM and 203.7 μM. By comparison, an IC50 value of 7 μM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 μM and 17.3 μM, r...

  2. Folylpolyglutamate synthetase: direct evidence for an acyl phosphate intermediate in the enzyme-catalyzed reaction

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

    The nature of the intermediate in the reaction catalyzed by folylpoly-..gamma..-glutamate synthetase (FPGS) has been investigated. Incubation of ..cap alpha..,..gamma..-(/sup 18/O)methotrexate with ATP, glutamate, and FPGS resulted in the formation of (/sup 18/O)phosphate, thus providing strong evidence for the formation of a ..gamma..-glutamyl phosphate during catalysis. The inorganic phosphate formed in the enzyme-catalyzed reaction was separated from other products and substrates by chromatography on DEAE-cellulose, then converted to the trimethyl ester, and analyzed by mass spectroscopy. Stoichiometric formation of (/sup 18/O)phosphate was observed in the case of the E. coli enzyme, isolated from a transformant containing the cloned FPGS-dihydrofolate synthetase (folC) gene. In addition, /sup 31/P-NMR analysis of the phosphate isolated from the reaction using E. coli FPGS showed the expected /sup 18/O-isotopic perturbations due to both singly bonded and doubly bonded P-/sup 18/O species. Similar experiments were carried out with FPGS isolated from hog liver. In this case, the small amounts of pure enzyme available precluded use of the NMR technique. However, mass spectral analysis of the derivatized phosphate product revealed the presence of (/sup 18/O)-trimethyl phosphate, thus indicating that the reaction catalyzed by the mammalian enzyme also proceeds via an acyl phosphate intermediate.

  3. 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.

  4. tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

    Science.gov (United States)

    Myers, Christopher A; Kuhla, Birte; Cusack, Stephen; Lambowitz, Alan M

    2002-03-05

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

  5. Nucleotide sequence and developmental expression of Acanthamoeba S-adenosylmethionine synthetase gene.

    Science.gov (United States)

    Ahn, K S; Henney, H R

    1997-03-20

    We have isolated and characterized a cDNA (cDNA1) from an Acanthamoeba cDNA library encoding the enzyme S-adenosylmethionine (SAM) synthetase (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6). The nucleotide sequence exhibits about 61-73% overall similarity to the corresponding gene of other organisms. The cDNA displays extreme codon bias with a preference for C or G in the third position. A putative initiation site and an ATP-binding site are identified. An amino acid content of 388 and a molecular mass of about 44,000 Daltons are deduced for the enzyme. Putative phosphorylation sites which might be involved in regulation of the enzyme are revealed. The cDNA was expressed in Escherichia coli BL21(DE3), and the identity of the protein product confirmed by Western blotting analysis. Northern analyses of the expression of the Acanthamoeba SAM synthetase gene during development revealed a pronounced reduction in the level of transcripts as amoebae converted to cysts.

  6. 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

  7. 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.

  8. Mammalian folylpoly-γ-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

    International Nuclear Information System (INIS)

    Cichowicz, D.J.; Shane, B.

    1987-01-01

    Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K + was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied

  9. 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.

  10. Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase.

    Science.gov (United States)

    Occhipinti, Andrea; Berlicki, Łukasz; Giberti, Samuele; Dziedzioła, Gabriela; Kafarski, Paweł; Forlani, Giuseppe

    2010-01-01

    Aiming at the rational design of new herbicides, the availability of the three-dimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry. Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non-competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity.

  11. 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.

  12. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Marta Spodenkiewicz

    2016-10-01

    Full Text Available Glutamine synthetase (GS is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  13. Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.

    Science.gov (United States)

    Ben-Salem, Salma; Gleeson, Joseph G; Al-Shamsi, Aisha M; Islam, Barira; Hertecant, Jozef; Ali, Bassam R; Al-Gazali, Lihadh

    2015-06-01

    Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.

  14. Mutations in the glutaminyl-tRNA synthetase gene cause early-onset epileptic encephalopathy.

    Science.gov (United States)

    Kodera, Hirofumi; Osaka, Hitoshi; Iai, Mizue; Aida, Noriko; Yamashita, Akio; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

    2015-02-01

    Aminoacylation is the process of attaching amino acids to their cognate tRNA, and thus is essential for the translation of mRNA into protein. This direct interaction of tRNA with amino acids is catalyzed by aminoacyl-tRNA synthetases. Using whole-exome sequencing, we identified compound heterozygous mutations [c.169T>C (p.Tyr57His) and c.1485dup (p.Lys496*)] in QARS, which encodes glutaminyl-tRNA synthetase, in two siblings with early-onset epileptic encephalopathy (EOEE). Recessive mutations in QARS, including the loss-of-function missense mutation p.Tyr57His, have been reported to cause intractable seizures with progressive microcephaly. The p.Lys496* mutation is novel and causes truncation of the QARS protein, leading to a deletion of part of the catalytic domain and the entire anticodon-binding domain. Transient expression of the p.Lys496* mutant in neuroblastoma 2A cells revealed diminished and aberrantly aggregated expression, indicating the loss-of-function nature of this mutant. Together with the previous report, our data suggest that abnormal aminoacylation is one of the underlying pathologies of EOEE.

  15. Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

    Science.gov (United States)

    Parker, J; Fishman, S E

    1979-01-01

    The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA. PMID:374370

  16. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Bernard, Stéphanie M.; Møller, Anders Laurell Blom; Dionisio, Giuseppe

    2008-01-01

    We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...

  17. Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

    Science.gov (United States)

    Parker, J; Fishman, S E

    1979-04-01

    The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA.

  18. Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects

    NARCIS (Netherlands)

    Santer, R.; Muhle, H.; Suormala, T.; Baumgartner, E. R.; Duran, M.; Yang, X.; Aoki, Y.; Suzuki, Y.; Stephani, U.

    2003-01-01

    We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the

  19. 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...

  20. 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

  1. Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

    Science.gov (United States)

    Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2003-01-01

    The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

  2. 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

  3. Genomic insights into the distribution, genetic diversity and evolution of polyketide synthases and nonribosomal peptide synthetases.

    Science.gov (United States)

    Wang, Hao; Sivonen, Kaarina; Fewer, David P

    2015-12-01

    Polyketides and nonribosomal peptides are important secondary metabolites that exhibit enormous structural diversity, have many pharmaceutical applications, and include a number of clinically important drugs. These complex metabolites are most commonly synthesized on enzymatic assembly lines of polyketide synthases and nonribosomal peptide synthetases. Genome-mining studies making use of the recent explosion in the number of genome sequences have demonstrated unexpected enzymatic diversity and greatly expanded the known distribution of these enzyme systems across the three domains of life. The wealth of data now available suggests that genome-mining efforts will uncover new natural products, novel biosynthetic mechanisms, and shed light on the origin and evolution of these important enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. De novo design and engineering of non-ribosomal peptide synthetases

    Science.gov (United States)

    Bozhüyük, Kenan A. J.; Fleischhacker, Florian; Linck, Annabell; Wesche, Frank; Tietze, Andreas; Niesert, Claus-Peter; Bode, Helge B.

    2018-03-01

    Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.

  5. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles...... in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

  6. Biosynthesis of the Peptide Antibiotic Feglymycin by a Linear Nonribosomal Peptide Synthetase Mechanism.

    Science.gov (United States)

    Gonsior, Melanie; Mühlenweg, Agnes; Tietzmann, Marcel; Rausch, Saskia; Poch, Annette; Süssmuth, Roderich D

    2015-12-01

    Feglymycin, a peptide antibiotic produced by Streptomyces sp. DSM 11171, consists mostly of nonproteinogenic phenylglycine-type amino acids. It possesses antibacterial activity against methicillin-resistant Staphylococcus aureus strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (feg) biosynthesis gene cluster, which codes for a 13-module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid l-Hpg proved the identity of the feg cluster. Feeding of Hpg-related unnatural amino acids was not successful. This characterization of the feg cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Sherry L. Mowbray

    2014-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  8. Inhibition of glutamine synthetase: a potential drug target in Mycobacterium tuberculosis.

    Science.gov (United States)

    Mowbray, Sherry L; Kathiravan, Muthu K; Pandey, Abhishek A; Odell, Luke R

    2014-08-26

    Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6-9 months) and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  9. 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

    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......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...... but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia....

  10. 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.

  11. 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

    the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial...... encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers...... 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...

  12. Editing mechanism of aminoacyl-tRNA synthetases operates by a hybrid ribozyme/protein catalyst.

    Science.gov (United States)

    Hagiwara, Yohsuke; Field, Martin J; Nureki, Osamu; Tateno, Masaru

    2010-03-03

    Aminoacyl-tRNA synthetases (aaRSs) are critical for the translational process, catalyzing the attachment of specific amino acids to their cognate tRNAs. To ensure formation of the correct aminoacyl-tRNA, and thereby enhance the reliability of translation, several aaRSs have an editing capability that hinders formation of misaminoacylated tRNAs. We investigated theoretically the mechanism of the editing reaction for a class I enzyme, leucyl-tRNA synthetase (LeuRS), complexed with a misaminoacylated tRNA(Leu), employing ab initio hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations. It is shown that the water molecule that acts as the nucleophile in the editing reaction is activated by a 3'-hydroxyl group at the 3'-end of tRNA(Leu) and that the O2' atom of the leaving group of the substrate is capped by one of the water's hydrogen atoms. Thus, it is shown that editing is a self-cleavage reaction of the tRNA and so it is the tRNA, and not the protein, that drives the reaction. The protein does, however, have an important stabilizing effect on some high-energy intermediates along the reaction path, which is more efficient than the ribozyme would be alone. This indicates that editing is achieved by a novel "hybrid ribozyme/protein catalyst". Analysis of existing experimental data and additional modeling shows that this ribozymal mechanism appears to be widespread, occurring in the ribosome as well as in other aaRSs. It also suggests transitional forms that could have played an important role in the RNA world hypothesis for the origin of life.

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

    Science.gov (United States)

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

    2004-03-01

    A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells. Copyright 2004 John Wiley & Sons, Ltd.

  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/Mg 2+ /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. Kinetic isotope effect studies of the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.; Parkin, D.W.; Schramm, V.L.

    1986-01-01

    S-adenosylmethionine (AdoMet) synthetase catalyzes a unique substitution reaction at the 5' carbon of MgATP. Kinetic isotope effect (V/K) measurements have been used to investigate the mechanism of AdoMet synthetase from E. coli. Changes in 3 H/ 14 C ratios when AdoMet is formed from a mixture of either ([5'- 14 C]ATP and [5'- 12 C,1'- 3 H]ATP) or ([5'- 3 H]ATP and [5'- 1 H,1'- 14 C]ATP) were examined. The effects of varying the concentrations of the co-substrate methionine and the monovalent cation activator K + were investigated. Substitution of 14 C for 12 C at the 5' position of ATP yields a primary V/K kinetic isotope effect ( 12 C/ 14 C) of 1.128 +/- 0.004 at low K + and methionine concentrations. The observed isotope effect diminishes slightly to 1.107 +/- 0.003 when both K + and methionine are present at saturating concentrations, suggesting that MgATP has only a low commitment to catalysis from at conditions near Vmax. No secondary V/K 3 H isotope effect from [5'- 3 H]ATP was detected ( 1 H/ 3 H) = 0.997 +/- 0.003. The magnitude of the primary 14 C isotope effect and the small secondary 3 H effect demonstrate that AdoMet synthesis occurs with a S/sub N/ 2 transition state which is symmetric with respect to the sulfur nucleophile and the departing tripolyphosphate group

  16. 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...

  17. 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...... the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator....

  18. 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.

  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. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections

    International Nuclear Information System (INIS)

    De Benedetti, A.; Pytel, B.A.; Baglioni, C.

    1987-01-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested

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

    Energy Technology Data Exchange (ETDEWEB)

    Handgretinger, R.; Bruchelt, G.; Kimmig, A.; Lang, P.; Daurer, B.; Dopfer, R.; Treuner, J.; Niethammer, D. (Children' s Univ. Hospital, Tuebingen (Germany, F.R.))

    1990-02-01

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

  3. β-Keto and β-hydroxyphosphonate analogs of biotin-5’-AMP are inhibitors of holocarboxylase synthetase

    Science.gov (United States)

    Sittiwong, Wantanee; Cordonier, Elizabeth L.; Zempleni, Janos; Dussault, Patrick H.

    2014-01-01

    Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5′-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 μM and 203.7 μM. By comparison, an IC50 value of 7 μM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 μM and 17.3 μM, respectively, are consistent with the IC50 results, and close to the Ki obtained for biotinol-5'-AMP (7 μM). The β-ketoP and β-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5'-AMP inhibited HLCS by a mixed mechanism. PMID:25466176

  4. β-Keto and β-hydroxyphosphonate analogs of biotin-5'-AMP are inhibitors of holocarboxylase synthetase.

    Science.gov (United States)

    Sittiwong, Wantanee; Cordonier, Elizabeth L; Zempleni, Janos; Dussault, Patrick H

    2014-12-15

    Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5'-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 μM and 203.7 μM. By comparison, an IC50 value of 7 μM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 μM and 17.3 μM, respectively, are consistent with the IC50 results, and close to the Ki obtained for biotinol-5'-AMP (7 μM). The β-ketoP and β-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5'-AMP inhibited HLCS by a mixed mechanism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. 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.

  6. Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress

    OpenAIRE

    Schug, Zachary T.; Peck, Barrie; Jones, Dylan T.; Zhang, Qifeng; Grosskurth, Shaun; Alam, Israt S.; Goodwin, Louise M.; Smethurst, Elizabeth; Mason, Susan; Blyth, Karen; McGarry, Lynn; James, Daniel; Shanks, Emma; Kalna, Gabriela; Saunders, Rebecca E.

    2015-01-01

    Summary A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and A...

  7. Protein S-Thiolation by Glutathionylspermidine (Gsp): THE ROLE OF ESCHERICHIA COLI Gsp SYNTHETASE/AMIDASE IN REDOX REGULATION*

    OpenAIRE

    Chiang, Bing-Yu; Chen, Tzu-Chieh; Pai, Chien-Hua; Chou, Chi-Chi; Chen, Hsuan-He; Ko, Tzu-Ping; Hsu, Wen-Hung; Chang, Chun-Yang; Wu, Whei-Fen; Wang, Andrew H.-J.; Lin, Chun-Hung

    2010-01-01

    Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification forme...

  8. 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...

  9. 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.

  10. 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.

  11. Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

    International Nuclear Information System (INIS)

    Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A. S.; Macedo-Ribeiro, Sandra

    2010-01-01

    The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

  12. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF-P ......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.......The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF...

  13. Toward understanding phosphoseryl-tRNACys formation: the crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase.

    Science.gov (United States)

    Kamtekar, Satwik; Hohn, Michael J; Park, Hee-Sung; Schnitzbauer, Michael; Sauerwald, Anselm; Söll, Dieter; Steitz, Thomas A

    2007-02-20

    A number of archaeal organisms generate Cys-tRNA(Cys) in a two-step pathway, first charging phosphoserine (Sep) onto tRNA(Cys) and subsequently converting it to Cys-tRNA(Cys). We have determined, at 3.2-A resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, alpha(4) synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (alphabeta)(2) phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.

  14. Toward Understanding Phosphoseryl-tRNA Cys Formation: The Crystal Structure of Methanococcus maripaludis Phosphoseryl-tRNA Synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Kamtekar ,S.; Hohn, M.; Park, h.; Schnitzbauer, M.; Sauerwald, A.; Soll, D.; Steitz, T.

    2007-01-01

    A number of archaeal organisms generate Cys-tRNA{sup Cys} in a two-step pathway, first charging phosphoserine (Sep) onto tRNA{sup Cys} and subsequently converting it to Cys-tRNA{sup Cys}. We have determined, at 3.2-{angstrom} resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, {alpha}{sub 4} synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric ({alpha}{beta}){sub 2} phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.

  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. 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.

  17. 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.

  18. The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production.

    Science.gov (United States)

    Ireland, C; Peekhaus, N; Lu, P; Sangari, R; Zhang, A; Masurekar, P; An, Z

    2008-06-01

    Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp(-)/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

  19. 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.

  20. 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.

  1. A case of severe glutathione synthetase deficiency with novel GSS mutations

    Directory of Open Access Journals (Sweden)

    H. Xia

    2018-01-01

    Full Text Available Glutathione synthetase deficiency (GSSD is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

  2. A case of severe glutathione synthetase deficiency with novel GSS mutations

    Science.gov (United States)

    Xia, H.; Ye, J.; Wang, L.; Zhu, J.; He, Z.

    2018-01-01

    Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old. PMID:29340523

  3. 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.

  4. Maintainance of specificity, information, and thermostability in thermophilic Bacillus sp. glutamine synthetase.

    Science.gov (United States)

    Wedler, F C; Hoffmann, F M; Kenney, R; Carfi, J

    1976-01-01

    Glutamine synthetase has been purified to homogeneity from B. subtilis (37 degrees) B. stearothermophilus (55 degrees), and B. caldolyticus (75 degrees). Those characteristics compared include size (6.0 +/- 0.3 X 10(5) daltons), quaternary structure (12 SU) amino acid content, substrate Km's and specificity for structural analogs, metal ion activation, number and kind of separate feedback modifier sites, and the complexity of modifier-substrate and modifier-modifier site interactions. Although the 37 degrees and 55 degrees systems are quite similar, the 75 degrees system shows important alterations in substrate specificity and modes of modifier action. Whereas at 37 degrees and 55 degrees AMP inhibits synergistically with amino acids (glycine, glutamine, histidine), the 75 degrees enzyme is inhibited directly by the products ADP, (which assumes the role of AMP) and glutamine, plus other ligands. Ligand binding domains are compared and found to be very different. Thermostabilization occurs by (a) protection by bound L-glutamate, (b) protein aggregation, (c) trends in the content of total polar residues, total Asx + Flx residues, the average hydrophobicity, and (d) disulfide bond cross-linking. Such studies provide insights to molecular evolution occurring with changes in environmental stress.

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

    Science.gov (United States)

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

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression. PMID:27009341

  6. 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.

  7. Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis.

    Science.gov (United States)

    Peat, Thomas S; Böttcher, Christine; Newman, Janet; Lucent, Del; Cowieson, Nathan; Davies, Christopher

    2012-11-01

    Auxins are important for plant growth and development, including the control of fruit ripening. Conjugation to amino acids by indole-3-acetic acid (IAA)-amido synthetases is an important part of auxin homeostasis. The structure of the auxin-conjugating Gretchen Hagen3-1 (GH3-1) enzyme from grapevine (Vitis vinifera), in complex with an inhibitor (adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate), is presented. Comparison with a previously published benzoate-conjugating enzyme from Arabidopsis thaliana indicates that grapevine GH3-1 has a highly similar domain structure and also undergoes a large conformational change during catalysis. Mutational analyses and structural comparisons with other proteins have identified residues likely to be involved in acyl group, amino acid, and ATP substrate binding. Vv GH3-1 is a monomer in solution and requires magnesium ions solely for the adenlyation reaction. Modeling of IAA and two synthetic auxins, benzothiazole-2-oxyacetic acid (BTOA) and 1-naphthaleneacetic acid (NAA), into the active site indicates that NAA and BTOA are likely to be poor substrates for this enzyme, confirming previous enzyme kinetic studies. This suggests a reason for the increased effectiveness of NAA and BTOA as auxins in planta and provides a tool for designing new and effective auxins.

  8. 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.

  9. Cancer association study of aminoacyl-tRNA synthetase signaling network in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Yong-Wan Kim

    Full Text Available Aminoacyl-tRNA synthetases (ARSs and ARS-interacting multifunctional proteins (AIMPs exhibit remarkable functional versatility beyond their catalytic activities in protein synthesis. Their non-canonical functions have been pathologically linked to cancers. Here we described our integrative genome-wide analysis of ARSs to show cancer-associated activities in glioblastoma multiforme (GBM, the most aggressive malignant primary brain tumor. We first selected 23 ARS/AIMPs (together referred to as ARSN, 124 cancer-associated druggable target genes (DTGs and 404 protein-protein interactors (PPIs of ARSs using NCI's cancer gene index. 254 GBM affymetrix microarray data in The Cancer Genome Atlas (TCGA were used to identify the probe sets whose expression were most strongly correlated with survival (Kaplan-Meier plots versus survival times, log-rank t-test <0.05. The analysis identified 122 probe sets as survival signatures, including 5 of ARSN (VARS, QARS, CARS, NARS, FARS, and 115 of DTGs and PPIs (PARD3, RXRB, ATP5C1, HSP90AA1, CD44, THRA, TRAF2, KRT10, MED12, etc. Of note, 61 survival-related probes were differentially expressed in three different prognosis subgroups in GBM patients and showed correlation with established prognosis markers such as age and phenotypic molecular signatures. CARS and FARS also showed significantly higher association with different molecular networks in GBM patients. Taken together, our findings demonstrate evidence for an ARSN biology-dominant contribution in the biology of GBM.

  10. 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.

  11. 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.

  12. 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

  13. Characterization of the acetyl-CoA synthetase of Acetobacter aceti.

    Science.gov (United States)

    O'Sullivan, J; Ettlinger, L

    1976-12-20

    The acetate activating system of Acetobacter aceti has been studied. The enzyme responsible, acetyl-CoA synthetase, has been purified about 500-fold from crude cell extracts and was approximately 85% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The purified enzyme showed optimal activity at pH 7.6 in both Tris-HCL and potassium phosphate buffers. In its purest form, the enzyme was stable at 4 degrees-C but denatured upon freezing. The Km values for CoA, ATP and acetate were found to be 0.104 mM, 0.36 mM and 0.25 mM respectively; propionate and acrylate were also activated by the enzyme but not butyrate, isobutyrate or valerate. GTP, UTP, CTP and ADP could not replace ATP in the reaction, and cysteine or pantetheine failed to replace CoA. The cationic requirements were studied and of the divalent cations tested, only Mn2+ could significantly replace Mg2+ in the reaction; K+ and NH4+ stimulated enzyme activity but inhibited at high concentrations; Na+ was a poor activator, but did not inhibit at higher concentrations. The effect of a number of glucose and other metabolites on enzyme activity has been tested.

  14. 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.

  15. 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.

  16. Hypophysectomy decrease and growth hormone increases the turnover and mass of rat liver glutamine synthetase

    International Nuclear Information System (INIS)

    Lin, Chingkow; Dunn, A.

    1989-01-01

    Hypophysectomy diminishes rat liver glutamine synthetase (GS) activity and growth hormone (GH) administration restores this activity to normal levels; brain GS is unaffected. We have now investigated the effects of long-term hypophysectomy (45-day) and GH treatment on the GS mass (amount of enzyme) and turnover in rat liver and brain. Labeled GS was isolated by immunoprecipitation at intervals between one and six days after pulse administration of [U- 14 C] leucine and the GS half-life (t 1/2 ) was determined. The GS mass was obtained by immunoassay and by calculation using the specific activity of purified GS. GS turnover was calculated by multiplying the GS mass by the first-order rate constant of degradation (k d ). During the time course of each experiment, the GS mass did not change, indicating that in each o the three hormonal states studied, a steady state existed. Hypophysectomy increased the t 1/2 of hepatic GS from 3.8 to 8.8 days and decreased GS turnover from 0.38 to 0.1 μg/100 g body wt/day; the GH regimen used restored the turnover to above normal levels, 0.6μg/100 g body wt/day. The GS mass decreased from 2.0 to 1.2 μg/100 g body wt and GH restored the GS mass to normal levels. The brain enzyme was not affected by hypophysectomy or GH

  17. NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.

    Science.gov (United States)

    Symersky, Jindrich; Devedjiev, Yancho; Moore, Karen; Brouillette, Christie; DeLucas, Larry

    2002-07-01

    The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5. The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63%. Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions. The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-atom positions of the phosphoester group of the reaction intermediate. The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule. A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase.

    Science.gov (United States)

    Zhu, Y L; Pilon-Smits, E A; Tarun, A S; Weber, S U; Jouanin, L; Terry, N

    1999-12-01

    To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.

  4. Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase1

    Science.gov (United States)

    Zhu, Yong Liang; Pilon-Smits, Elizabeth A.H.; Tarun, Alice S.; Weber, Stefan U.; Jouanin, Lise; Terry, Norman

    1999-01-01

    To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity. PMID:10594104

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

    Science.gov (United States)

    Ottesen, Elizabeth A; Leadbetter, Jared R

    2011-05-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2014-03-01

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

  8. Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom).

    Science.gov (United States)

    Scarpeci, Telma E; Marro, Martin L; Bortolotti, Santiago; Boggio, Silvana B; Valle, Estela M

    2007-02-01

    Tomato (Solanum lycopersicum) fruit ripening implies that chloroplastic proteins are degraded and new proteins are synthesized. Supplementary nutrition is frequently required when tomato plants begin to fruit and continues until the end of the plant's life cycle. Ammonium assimilation is crucial in these fruit maturation and ripening processes. Glutamine synthetase (GS; EC 6.3.1.2), the main ammonium-fixing enzyme in plants, could not be detected in red fruits of several tomato varieties when growing under standard nutrition. In this paper, we analyze the influence of the nutritional status on the ammonium assimilation capacity of ripe tomato (cv. Micro-Tom) fruit. For this purpose, GS expression and protein profiles were followed in mature green and red fruits harvested from plants grown under standard or supplemented nutrition. Under standard nutrient regime (weekly supplied with 0.5 x Hoagland solution) GS activity was found in chloroplasts (GS2) of mature green fruits, but it was not detected either in the chromoplasts or in the cytosol of red fruits. When plants were shifted to a supplemented nutritional regime (daily supplied with 0.5 x Hoagland solution), GS was found in red fruits. Also, cytosolic transcripts (gs1) preferentially accumulated in red fruits under high nutrition. These results indicate that mature green Micro-Tom fruits assimilate ammonia through GS2 under standard nutrition, while ripe red fruits accumulate GS1 under high nutrition, probably in order to assimilate the extra N-compounds made available through supplemented nutrition.

  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. 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.

  11. 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.

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

    Science.gov (United States)

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

    2014-10-01

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

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

    International Nuclear Information System (INIS)

    Hickey, E.W.; Hirshfield, I.

    1987-01-01

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

  14. Arabidopsis plastidial folylpolyglutamate synthetase is required for seed reserve accumulation and seedling establishment in darkness.

    Directory of Open Access Journals (Sweden)

    Hongyan Meng

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

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

    Science.gov (United States)

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

    2016-05-01

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

  16. Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

    Science.gov (United States)

    Faghih, Omeed; Zhang, Zhongsheng; Ranade, Ranae M; Gillespie, J Robert; Creason, Sharon A; Huang, Wenlin; Shibata, Sayaka; Barros-Álvarez, Ximena; Verlinde, Christophe L M J; Hol, Wim G J; Fan, Erkang; Buckner, Frederick S

    2017-11-01

    Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus , Enterococcus , and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development. Copyright © 2017 American Society for Microbiology.

  17. Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

    Directory of Open Access Journals (Sweden)

    Rouhiainen Leo

    2008-09-01

    Full Text Available Abstract Background Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy. mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer. Results A comparative analysis of the adenylation domains from the first module of McyB (McyB1 and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants. Conclusion We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may

  18. Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel).

    Science.gov (United States)

    Zhang, Meng-Yi; Wei, Dong; Li, Ran; Jia, Hong-Ting; Liu, Yu-Wei; Taning, Clauvis Nji Tizi; Wang, Jin-Jun; Smagghe, Guy

    2018-04-01

    In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management. © 2018 Wiley Periodicals, Inc.

  19. Dual Organellar Targeting of Aminoacyl-tRNA Synthetases in Diatoms and Cryptophytes.

    Science.gov (United States)

    Gile, Gillian H; Moog, Daniel; Slamovits, Claudio H; Maier, Uwe-G; Archibald, John M

    2015-05-20

    The internal compartmentation of eukaryotic cells not only allows separation of biochemical processes but it also creates the requirement for systems that can selectively transport proteins across the membrane boundaries. Although most proteins function in a single subcellular compartment, many are able to enter two or more compartments, a phenomenon known as dual or multiple targeting. The aminoacyl-tRNA synthetases (aaRSs), which catalyze the ligation of tRNAs to their cognate amino acids, are particularly prone to functioning in multiple subcellular compartments. They are essential for translation, so they are required in every compartment where translation takes place. In diatoms, there are three such compartments, the plastid, the mitochondrion, and the cytosol. In cryptophytes, translation also takes place in the periplastid compartment (PPC), which is the reduced cytoplasm of the plastid's red algal ancestor and which retains a reduced red algal nucleus. We searched the organelle and nuclear genomes of the cryptophyte Guillardia theta and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana for aaRS genes and found an insufficient number of genes to provide each compartment with a complete set of aaRSs. We therefore inferred, with support from localization predictions, that many aaRSs are dual targeted. We tested four of the predicted dual targeted aaRSs with green fluorescent protein fusion localizations in P. tricornutum and found evidence for dual targeting to the mitochondrion and plastid in P. tricornutum and G. theta, and indications for dual targeting to the PPC and cytosol in G. theta. This is the first report of dual targeting in diatoms or cryptophytes. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rydzak, Thomas [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Garcia, David [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Stevenson, David M. [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Sladek, Margaret [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Klingeman, Dawn M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Holwerda, Evert K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Amador-Noguez, Daniel [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Brown, Steven D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center

    2017-05-01

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

  1. Mammalian folylpoly-γ-glutamate synthetase. 2. Substrate specificity and kinetic properties

    International Nuclear Information System (INIS)

    Cichowicz, D.J.; Shane, B.

    1987-01-01

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

  2. Poly(Adp-ribose) synthetase inhibition prevents lipopolysaccharide-induced peroxynitrite mediated damage in diaphragm.

    Science.gov (United States)

    Ozdülger, Ali; Cinel, Ismail; Unlü, Ali; Cinel, Leyla; Mavioglu, Ilhan; Tamer, Lülüfer; Atik, Ugur; Oral, Ugur

    2002-07-01

    Although the precise mechanism by which sepsis causes impairment of respiratory muscle contractility has not been fully elucidated, oxygen-derived free radicals are thought to play an important role. In our experimental study, the effects of poly(ADP-ribose) synthetase (PARS) inhibition on the diaphragmatic Ca(2+)-ATPase, malondialdehyde (MDA), and 3-nitrotyrosine (3-NT) levels and additionally histopathology of the diaphragm in lipopolysaccharide (LPS)-induced endotoxemia are investigated.Thirty-two male Wistar rats, weighing between 180-200 g were randomly divided into four groups. The first group (control; n=8) received saline solution and the second (LPS group; n=8) 10 mgkg(-1) LPS i.p. 3-Aminobenzamide (3-AB) as a PARS inhibitor; was given to the third group (C+3-AB, n=8) 20 min before administration of saline solution while the fourth group (LPS+3-AB, n=8) received 3-AB 20 min before LPS injection. Six hours later, under ketamin/xylasine anesthesia diapraghmatic specimens were obtained and the rats were decapitated. Diaphragmatic specimens were divided into four parts, three for biochemical analyses and one for histopathologic assessment. In the LPS group, tissue Ca(2+)-ATPase levels were found to be decreased and tissue MDA and 3-NT levels were found to be increased (P<0.05). In the LPS+3-AB group, 3-AB pretreatment inhibited the increase in MDA and 3-NT levels and Ca(2+)-ATPase activity remained similar to those in the control group (P<0.05). Histopathologic examination of diaphragm showed edema between muscle fibers only in LPS group. PARS inhibition with 3-AB prevented not only lipid peroxidation but also the decrease of Ca(2+)-ATPase activity in endotoxemia. These results highlights the importance of nitric oxide (NO)-peroxynitrite (ONOO(-))-PARS pathway in preventing free radical mediated injury. PARS inhibitors should further be investigated as a new thearapetic alternative in sepsis treatment.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

    Science.gov (United States)

    Quadri, L E; Weinreb, P H; Lei, M; Nakano, M M; Zuber, P; Walsh, C T

    1998-02-10

    The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

  6. Mutations in QARS, Encoding Glutaminyl-tRNA Synthetase, Cause Progressive Microcephaly, Cerebral-Cerebellar Atrophy, and Intractable Seizures

    Science.gov (United States)

    Zhang, Xiaochang; Ling, Jiqiang; Barcia, Giulia; Jing, Lili; Wu, Jiang; Barry, Brenda J.; Mochida, Ganeshwaran H.; Hill, R. Sean; Weimer, Jill M.; Stein, Quinn; Poduri, Annapurna; Partlow, Jennifer N.; Ville, Dorothée; Dulac, Olivier; Yu, Tim W.; Lam, Anh-Thu N.; Servattalab, Sarah; Rodriguez, Jacqueline; Boddaert, Nathalie; Munnich, Arnold; Colleaux, Laurence; Zon, Leonard I.; Söll, Dieter; Walsh, Christopher A.; Nabbout, Rima

    2014-01-01

    Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders. PMID:24656866

  7. 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

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Buddha,M.; Crane, B.

    2005-01-01

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

  10. 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...

  11. 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......, our data demonstrate that alpha-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity, although mutant forms of alpha-synuclein fail to restore this activity....

  12. 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

  13. 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...

  14. 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

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

    Directory of Open Access Journals (Sweden)

    Fengguang eGuo

    2015-07-01

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

  16. 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.

  17. Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

    Science.gov (United States)

    Lu, Jiongming; Bergert, Martin; Walther, Anita; Suter, Beat

    2014-11-27

    Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

  18. Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

    International Nuclear Information System (INIS)

    Wang, Xiaoying; Akasaka, Ryogo; Takemoto, Chie; Morita, Satoshi; Yamaguchi, Machiko; Terada, Takaho; Shirozu, Mikako; Yokoyama, Shigeyuki; Chen, Shilin; Si, Shuyi; Xie, Yong

    2011-01-01

    A hyperthermophilic adenylosuccinate synthetase from P. horikoshii OT3, which is 90–120 amino acids shorter than those from the vast majority of organisms, was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution. Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3 2 12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å 3 Da −1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution

  19. 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.

  20. 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 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...... on the substrate concentrations used. At excess acetyl-CoA to malonyl-CoA, greater amounts of acetyl-CoA were incorporated than theoretically expected from the malonyl-CoA pathway. At excess malonyl-CoA, less acetyl-CoA was incorporated than theoretically expected. - 4. An increase in the chain-length of fatty...

  1. Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

    Science.gov (United States)

    Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

    2015-04-01

    Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. 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....... The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters...... such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The K-m values for the three precursor substrates La-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 mu M respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM Mg...

  3. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600

    Directory of Open Access Journals (Sweden)

    Biaosheng Lin

    2016-01-01

    Full Text Available To clone and express the γ-polyglutamic acid (γ-PGA synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.

  4. Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development

    Science.gov (United States)

    Lu, Jiongming; Bergert, Martin; Walther, Anita; Suter, Beat

    2014-01-01

    Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases. PMID:25427601

  5. Overexpression of S-adenosyl-L-methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism.

    Science.gov (United States)

    Gong, Biao; Li, Xiu; VandenLangenberg, Kyle M; Wen, Dan; Sun, Shasha; Wei, Min; Li, Yan; Yang, Fengjuan; Shi, Qinghua; Wang, Xiufeng

    2014-08-01

    S-adenosyl-L-methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1-overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na(+) absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. 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.

  7. Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

    Czech Academy of Sciences Publication Activity Database

    Bakal, Tomáš; Goo, K.-S.; Najmanová, Lucie; Plháčková, Kamila; Kadlčík, Stanislav; Ulanová, Dana

    2015-01-01

    Roč. 108, č. 5 (2015), s. 1267-1274 ISSN 0003-6072 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Nonribosomal peptide synthetase * Adenylation domain * Actinomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 1.944, year: 2015

  8. 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.

  9. Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

    1983-01-01

    A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

  10. The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit

    NARCIS (Netherlands)

    Schoneveld, Onard J. L. M.; Gaemers, Ingrid C.; Hoogenkamp, Maarten; Lamers, Wouter H.

    2005-01-01

    As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When

  11. 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

    -fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent...

  12. Up-regulation of asparagine synthetase expression is not linked to the clinical response to L-asparaginase in pediatric acute lymphoblastic leukemia

    NARCIS (Netherlands)

    I.M. Appel (Inge); M.L. den Boer (Monique); J.P.P. Meijerink (Jules); A.J.P. Veerman (Anjo); N.C.M. Reniers (N. C M); R. Pieters (Rob)

    2006-01-01

    textabstractL-asparaginase (L-Asp) is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Asparagine synthetase (AS) opposes the action of L-Asp by resynthesis

  13. Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase

    Science.gov (United States)

    Tang, Yi; Wang, Pin; Van Deventer, James A.; Link, A. James; Tirrell, David A.

    2011-01-01

    A leucine analog containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated. PMID:19670197

  14. 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

  15. Assignment of two human autoantigen genes-isoleucyl-tRNA synthetase locates to 9q21 and lysyl-tRNA synthetase locates to 16q23-q24

    Energy Technology Data Exchange (ETDEWEB)

    Nichols, R.C.; Blinder, J.; Pai, S.I. [National Inst. of Health, Bethesda, MD (United States)] [and others

    1996-08-15

    Protein synthesis is initiated by the attachment of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases (aaRS). Five of twenty human aaRS (histidyl-RS, threonyl-RS, alanyl-RS, glycyl-RS, and isoleucyl-RS) have been identified as targets of autoantibodies in the autoimmune disease polymyositis/dermatomyositis. Autoantibodies to human lysyl-RS, a sixth autoantigenic aminoacyl-RS, were recently identified. The genes for histidyl-RS and threonyl-RS have been localized to chromosome 5, and we recently reported that the genes for alanyl-RS and glycyl-RS localize to chromosomes 16 and 7, respectively. To understand the genesis of autoimmune responses to aaRS better, we have used PCR-based screening of somatic cell hybrid panels and fluorescence in situ hybridization (FISH) to assign the genes for isoleucyl-RS and lysyl-RS. 19 refs., 1 fig.

  16. 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

  17. Crystallization and preliminary X-ray diffraction analysis of selenophosphate synthetases from Trypanosoma brucei and Leishmania major.

    Science.gov (United States)

    Faim, Lívia Maria; Rosa e Silva, Ivan; Bertacine Dias, Marcio Vinicius; D'Muniz Pereira, Humberto; Brandao-Neto, José; Alves da Silva, Marco Túlio; Thiemann, Otavio Henrique

    2013-08-01

    Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.

  18. Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.

    Science.gov (United States)

    Schug, Zachary T; Peck, Barrie; Jones, Dylan T; Zhang, Qifeng; Grosskurth, Shaun; Alam, Israt S; Goodwin, Louise M; Smethurst, Elizabeth; Mason, Susan; Blyth, Karen; McGarry, Lynn; James, Daniel; Shanks, Emma; Kalna, Gabriela; Saunders, Rebecca E; Jiang, Ming; Howell, Michael; Lassailly, Francois; Thin, May Zaw; Spencer-Dene, Bradley; Stamp, Gordon; van den Broek, Niels J F; Mackay, Gillian; Bulusu, Vinay; Kamphorst, Jurre J; Tardito, Saverio; Strachan, David; Harris, Adrian L; Aboagye, Eric O; Critchlow, Susan E; Wakelam, Michael J O; Schulze, Almut; Gottlieb, Eyal

    2015-01-12

    A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis.

    Science.gov (United States)

    Rai, A N; Lindblad, P; Bergman, B

    1986-11-01

    Using the ammonium analogue (14)CH3NH 3 (+) , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-DL-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (ΔΨ), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous (14)CH3NH 3 (+) or NH 4 (+) because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.

  20. The putative tRNA 2-thiouridine synthetase Ncs6 is an essential sulfur carrier in Methanococcus maripaludis.

    Science.gov (United States)

    Liu, Yuchen; Long, Feng; Wang, Liangliang; Söll, Dieter; Whitman, William B

    2014-03-18

    Thiolation of carbon-2 of uridine located in the first position of the anticodons of tRNAUUG(Gln), tRNAUUC(Glu), and tRNAUUU(Lys) is a conserved RNA modification event requiring the 2-thiouridine synthetase Ncs6/Ctu1 in archaea and eukaryotes. Ncs6/Ctu1 activates uridine by adenylation, but its role in sulfur transfer is unclear. Here we show that Mmp1356, the Ncs6/Ctu1 homolog in the archaeon Methanococcus maripaludis, forms a persulfide enzyme adduct with an active site cysteine; this suggests that Mmp1356 directly participates in sulfur transfer as a persulfide carrier. Transposon mutagenesis shows that Mmp1356 is likely to be an essential protein. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP

    DEFF Research Database (Denmark)

    Manav, Melek Cemre; Beljantseva, Jelena; Bojer, Martin Saxtorph

    2018-01-01

    '-diphosphate ([p]ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, Staphylococcus aureus, encodes three RSH enzymes: a multi-domain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, Rel......Q (SAS1) and RelP (SAS2). In Bacillus subtilis, RelQ (SAS1) was shown to form a tetramer that is activated by pppGpp and inhibited by single stranded RNA, but the structural and functional regulation of RelP (SAS2) is unexplored. Here, we present crystal structures of S. aureus RelP in two major...

  2. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

    DEFF Research Database (Denmark)

    Noh, Soo Min; Shin, Seunghyeon; Min Lee, Gyun

    2018-01-01

    and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated......To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1...... in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation...

  3. Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene

    DEFF Research Database (Denmark)

    hart, Leen M; Hansen, Torben; Rietveld, Ingrid

    2005-01-01

    Previously, we have shown that a mutation in the mitochondrial DNA-encoded tRNA(Leu(UUR)) gene is associated with type 2 diabetes. One of the consequences of this mutation is a reduced aminoacylation of tRNA(Leu(UUR)). In this study, we have examined whether variants in the leucyl tRNA synthetase...... gene (LARS2), involved in aminoacylation of tRNA(Leu(UUR)), associate with type 2 diabetes. Direct sequencing of LARS2 cDNA from 25 type 2 diabetic subjects revealed eight single nucleotide polymorphisms. Two of the variants were examined in 7,836 subjects from four independent populations...... no significant differences in clinical variables between carriers and noncarriers. In this study, we provide evidence that the LARS2 gene may represent a novel type 2 diabetes susceptibility gene. The mechanism by which the H324Q variant enhances type 2 diabetes risk needs to be further established...

  4. Structure Elucidation and Activity of Kolossin A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

    Science.gov (United States)

    Bode, Helge B; Brachmann, Alexander O; Jadhav, Kirtikumar B; Seyfarth, Lydia; Dauth, Christina; Fuchs, Sebastian W; Kaiser, Marcel; Waterfield, Nick R; Sack, Holger; Heinemann, Stefan H; Arndt, Hans-Dieter

    2015-08-24

    The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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.

  6. Effect of 60Co γ radiation on the valyl-tRNA synthetase isolated from chick embryo brain

    International Nuclear Information System (INIS)

    Boloni, E.; Szabo, L.D.

    1978-01-01

    he effect of 60 Coγ irradiation on the activity of valyl-tRNA synthetase isolated from chick embryo brain was studied. The enzyme activity exponentially decreased in the dose range 10 to 200 krad. The first step of the enzyme action, i.e. amino acid activation, was found to be less sensitive to irradiation than the whole reaction, the formation of valyl-tRNA. 2-Mercapto ethanol and/or glycerol had a significant radioprotective effect. The lesion caused by radiation in the enzyme was also influenced by its concentration during exposure (diluted effect). According to gel-electrophoretic experiments, no chain rupture occurred in the enzyme molecule. Not even a change in Ksub(m) was observed; however, the maximum velocity of the reaction was found to decrease with increasing radiation dose. (author)

  7. A new mutation in the gene encoding mitochondrial seryl-tRNA synthetase as a cause of HUPRA syndrome.

    Science.gov (United States)

    Rivera, Henry; Martín-Hernández, Elena; Delmiro, Aitor; García-Silva, María Teresa; Quijada-Fraile, Pilar; Muley, Rafael; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

    2013-09-13

    HUPRA syndrome is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis. This syndrome was previously described in three patients with a homozygous mutation c.1169A > G (p.D390G) in SARS2, encoding the mitochondrial seryl-tRNA synthetase. Here we report the clinical and genetic findings in a girl and her brother. Both patients were clinically diagnosed with the HUPRA syndrome. Analysis of the pedigree identified a new homozygous mutation c.1205G > A (p.R402H) in SARS2 gene. This mutation is very rare in the population and it is located at the C-terminal globular domain of the homodimeric enzyme very close to p.D390G. Several data support that p.R402H mutation in SARS2 is a new cause of HUPRA syndrome.

  8. Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii

    International Nuclear Information System (INIS)

    Fukunaga, Ryuya; Ishitani, Ryuichiro; Nureki, Osamu; Yokoyama, Shigeyuki

    2004-01-01

    The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. All five tRNA Leu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNA Leu , suggesting that they are LeuRS–tRNA Leu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNA Leu , with a corresponding crystal volume per protein weight of 2.9 Å 3 Da −1 and a solvent content of 57.3%

  9. Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA

    International Nuclear Information System (INIS)

    Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S.

    1987-01-01

    Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the α 2 β 2 enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[ 14 C]tRNA/sub ox//sup Phe/ covalent complex indicates that the large (α, M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The [ 14 C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the α subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases

  10. Substrate specificity and catalysis by the editing active site of alanyl-tRNA synthetase from Escherichia coli†

    Science.gov (United States)

    Pasman, Zvi; Robey-Bond, Susan; Mirando, Adam C.; Smith, Gregory J.; Lague, Astrid; Francklyn, Christopher S.

    2011-01-01

    Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free standing P. horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNAAla and Ala-tRNAAla as substrates, the deacylation activities of the wild type and five different E. coli AlaRS editing site substitution mutants were characterized. The wild type AlaRS editing domain deacylated Ser-tRNAAla with a kcat/KM of 6.6 × 105 M−1 s−1, equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation, but only 12.2-fold greater than the rate with Ala-tRNAAla. While the E664A and T567G substitutions only minimally decreased kcat/KM, Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in kcat/KM in the range of 6-, 7.3-, and 15-fold. C666A AlaRS was 1.4-fold more active on Ala-tRNAAla relative to Ser-tRNAAla, providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine mis-incorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNAAla. PMID:21241052

  11. 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.

  12. 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.

  13. Immunological recovery and dose evaluation in IFN-alpha treatment of hairy cell leukemia: analysis of leukocyte differentiation antigens, NK and 2',5'-oligoadenylate synthetase activity

    DEFF Research Database (Denmark)

    Nielsen, B; Hokland, M; Justesen, J

    1989-01-01

    , the number of NK cells normalized in 90 to 180 d, whereas normalization of B cell number was seen only after 180 to 360 d of treatment. Mean pretreatment 2-5A synthetase activity was normal or low, but upon treatment the levels rose immediately to higher than normal values and remained high throughout......A low-dose interferon (IFN)-alpha regimen for the treatment of hairy cell leukemia (HCL) was evaluated by following changes in leukocyte differentiation antigens (LDA), natural killer cell (NK) and 2',5'-oligoadenylate (2-5A) synthetase activities. Due to hairy cells' (HC) weak expression...... of several antigens positive for T cells, B cells, NK cells and monocytes, the use of a double marker specific for hairy cells was needed to distinguish the different subpopulations. Analysis of LDA in peripheral blood (PB) showed a total normalization of the T cell and monocyte numbers within 90 days...

  14. 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

    , and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[ß,¿-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can...... of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine...... of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the ¿-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining...

  15. 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.

  16. 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

    The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2',3'-dialdehyde ATP (oATP). The reaction displayed complex saturation kinetics with respect to oATP with an apparent KD of approximately 0.8 m...... of enzymatic activity. These results imply a functional role for at least two of the identified amino acid residues....

  17. Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model: Mechanism for deficient glutamatergic transmission?

    Czech Academy of Sciences Publication Activity Database

    Olabarria, M.; Noristani, H. N.; Verkhratsky, Alexei; Rodríguez Arellano, Jose Julio

    2011-01-01

    Roč. 6, č. 1 (2011), s. 55-63 ISSN 1750-1326 R&D Projects: GA ČR GA309/09/1696; GA ČR(CZ) GAP304/11/0184; GA ČR GA305/08/1384; GA ČR GA309/08/1381 Institutional research plan: CEZ:AV0Z50390703 Keywords : astroglia * glutamine synthetase * Alzheimer ?'?s disease Subject RIV: FH - Neurology Impact factor: 4.278, year: 2011

  18. 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.

  19. Improved stress tolerance and productivity in transgenic rice plants constitutively expressing the Oryza sativa glutathione synthetase OsGS under paddy field conditions.

    Science.gov (United States)

    Park, Seong-Im; Kim, Young-Saeng; Kim, Jin-Ju; Mok, Ji-Eun; Kim, Yul-Ho; Park, Hyang-Mi; Kim, Il-Sup; Yoon, Ho-Sung

    2017-08-01

    Reactive oxygen species, which increase under various environmental stresses, have deleterious effects on plants. An important antioxidant, glutathione, is used to detoxify reactive oxygen species in plant cells and is mainly produced by two enzymes: gamma-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the glutathione synthetase gene (OsGS) in rice, we generated four independent transgenic rice plants (TG1-TG4) that overexpressed OsGS under the control of the constitutively expressed OsCc1 promoter. When grown under natural paddy field conditions, the TG rice plants exhibited greater growth development, higher chlorophyll content, and higher GSH/GSSH ratios than control wild-type (WT) rice plants. Subsequently, the TG rice plants enhanced redox homeostasis by preventing hydroperoxide-mediated membrane damage, which improved their adaptation to environmental stresses. As a result, TG rice plants improved rice grain yield and total biomass following increases in panicle number and number of spikelets per panicle, despite differences in climate during the cultivation periods of 2014 and 2015. Overall, our results indicate that OsGS overexpression improved redox homeostasis by enhancing the glutathione pool, which resulted in greater tolerance to environmental stresses in the paddy fields. Copyright © 2017. Published by Elsevier GmbH.

  20. Long-Range Structural Effects of a Charcot-Marie-Tooth Disease-Causing Mutation in Human Glycyl-TRNA Synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Xie, W.; Nangle, L.A.; Zhang, W.; Schimmel, P.; Yang, X.-L.

    2009-06-04

    Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located {approx}30 {angstrom} away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.

  1. 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.

  2. 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.

  3. Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays)

    Science.gov (United States)

    2005-01-01

    Unlike animals which accumulate glutathione (γ-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or γ-glutamyl-L-cysteinyl-β-alanine)], wheat (hydroxymethylglutathione or γ-glutamyl-L-cysteinyl-serine) and maize (γ-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into γ-Glu-Cys. The soya bean enzyme primarily incorporated β-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and α-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor. PMID:16008521

  4. 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.

  5. Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

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    Stefanie Berger

    2012-01-01

    Full Text Available The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM=0.27±0.05 mM that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell.

  6. A Conserved Proline Triplet in Val-tRNA Synthetase and the Origin of Elongation Factor P

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    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.

  7. Crystal Structure of an Indole-3-Acetic Acid Amido Synthetase from Grapevine Involved in Auxin Homeostasis[W

    Science.gov (United States)

    Peat, Thomas S.; Böttcher, Christine; Newman, Janet; Lucent, Del; Cowieson, Nathan; Davies, Christopher

    2012-01-01

    Auxins are important for plant growth and development, including the control of fruit ripening. Conjugation to amino acids by indole-3-acetic acid (IAA)-amido synthetases is an important part of auxin homeostasis. The structure of the auxin-conjugating Gretchen Hagen3-1 (GH3-1) enzyme from grapevine (Vitis vinifera), in complex with an inhibitor (adenosine-5′-[2-(1H-indol-3-yl)ethyl]phosphate), is presented. Comparison with a previously published benzoate-conjugating enzyme from Arabidopsis thaliana indicates that grapevine GH3-1 has a highly similar domain structure and also undergoes a large conformational change during catalysis. Mutational analyses and structural comparisons with other proteins have identified residues likely to be involved in acyl group, amino acid, and ATP substrate binding. Vv GH3-1 is a monomer in solution and requires magnesium ions solely for the adenlyation reaction. Modeling of IAA and two synthetic auxins, benzothiazole-2-oxyacetic acid (BTOA) and 1-naphthaleneacetic acid (NAA), into the active site indicates that NAA and BTOA are likely to be poor substrates for this enzyme, confirming previous enzyme kinetic studies. This suggests a reason for the increased effectiveness of NAA and BTOA as auxins in planta and provides a tool for designing new and effective auxins. PMID:23136372

  8. 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.

  9. Functional substitution of a eukaryotic glycyl-tRNA synthetase with an evolutionarily unrelated bacterial cognate enzyme.

    Directory of Open Access Journals (Sweden)

    Chin-I Chien

    Full Text Available Two oligomeric types of glycyl-tRNA synthetase (GlyRS are found in nature: a α2 type and a α2β2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α2-type enzymes can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1- strain, while Escherichia coli GlyRS (a α2β2-type enzyme and A. thaliana organellar GlyRS (a (αβ2-type enzyme failed to rescue either defect of the yeast mull allele. However, a head-to-tail αβ fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α2-type eukaryotic GlyRS may be functionally substituted with a α2β2-type bacterial cognate enzyme despite their remote evolutionary relationships.

  10. 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.

  11. 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.

  12. Targeting CPS1 in the treatment of Carbamoyl phosphate synthetase 1 (CPS1) deficiency, a urea cycle disorder.

    Science.gov (United States)

    Diez-Fernandez, Carmen; Häberle, Johannes

    2017-04-01

    Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder (UCD), which can lead to life-threatening hyperammonemia. Unless promptly treated, it can result in encephalopathy, coma and death, or intellectual disability in surviving patients. Over recent decades, therapies for CPS1D have barely improved leaving the management of these patients largely unchanged. Additionally, in many cases, current management (protein-restriction and supplementation with citrulline and/or arginine and ammonia scavengers) is insufficient for achieving metabolic stability, highlighting the importance of developing alternative therapeutic approaches. Areas covered: After describing UCDs and CPS1D, we give an overview of the structure- function of CPS1. We then describe current management and potential novel treatments including N-carbamoyl-L-glutamate (NCG), pharmacological chaperones, and gene therapy to treat hyperammonemia. Expert opinion: Probably, the first novel CPS1D therapies to reach the clinics will be the already commercial substance NCG, which is the standard treatment for N-acetylglutamate synthase deficiency and has been proven to rescue specific CPS1D mutations. Pharmacological chaperones and gene therapy are under development too, but these two technologies still have key challenges to be overcome. In addition, current experimental therapies will hopefully add further treatment options.

  13. The Cytoplasmic Prolyl-tRNA Synthetase of the Malaria Parasite is a Dual-Stage Target for Drug Development

    Science.gov (United States)

    Herman, Jonathan D.; Pepper, Lauren R.; Cortese, Joseph F.; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R.; Ribacke, Ulf; Lukens, Amanda K.; Santos, Sofia A.; Patel, Vishal; Clish, Clary B.; Sullivan, William J.; Zhou, Huihao; Bopp, Selina E.; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M.; Keller, Tracy L.; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F.; Mazitschek, Ralph

    2015-01-01

    The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for the development of the next-generation of antimalarial drugs. Using an integrated chemogenomics approach that combined drug-resistance selection, whole genome sequencing and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivatives such as halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the P. berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is highly active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses, and represents a promising lead for the development of dual-stage next generation antimalarials. PMID:25995223

  14. Targeting Prolyl-tRNA Synthetase to Accelerate Drug Discovery against Malaria, Leishmaniasis, Toxoplasmosis, Cryptosporidiosis, and Coccidiosis.

    Science.gov (United States)

    Jain, Vitul; Yogavel, Manickam; Kikuchi, Haruhisa; Oshima, Yoshiteru; Hariguchi, Norimitsu; Matsumoto, Makoto; Goel, Preeti; Touquet, Bastien; Jumani, Rajiv S; Tacchini-Cottier, Fabienne; Harlos, Karl; Huston, Christopher D; Hakimi, Mohamed-Ali; Sharma, Amit

    2017-10-03

    Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. 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.

  16. 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.

  17. Modulation of astrocytic glutamine synthetase expression and cell viability by histamine in cultured cortical astrocytes exposed to OGD insults.

    Science.gov (United States)

    Wang, Xiao-Fen; Hu, Wei-Wei; Yan, Hai-Jing; Tan, Li; Gao, Jie-Qiong; Tian, Yue-Yang; Shi, Xiao-Jie; Hou, Wei-Wei; Li, Juan; Shen, Yao; Chen, Zhong

    2013-08-09

    Histamine, a neurotransmitter or neuromodulator has been demonstrated to be neuroprotective in cerebral ischemia. However, few reports concern its function on astrocytes during cerebral ischemia. The purpose of this study was to investigate the effects of histamine on astrocytic cell damage and glutamate signaling, especially on glutamine synthetase (GS) expression in primary cultured cortical astrocytes exposed to oxygen-glucose deprivation (OGD) insult. OGD for 6h caused a severe damage of astrocytic mitochondrial function, and decreased GS expression and then increased the extracellular glutamate level. Pretreatment with histamine significantly prevented the cell damage and rescued the expression of GS in a concentration-dependent manner. The protective effect of histamine on astrocytic cell damage could be partly reversed either by H1 receptor antagonist pyrilamine or H2 receptor antagonist cimetidine. However, the regulatory effect of histamine on GS expression was antagonized only by pyrilamine. In addition, bisindolylmaleimide II, a broad-spectrum inhibitor of PKC, reversed the regulatory action of histamine on GS expression. These results indicate that histamine can effectively protect against OGD-induced cell damage in astrocytes through H1 and H2 receptors, and its regulatory effect on astrocytic GS expression may be due to the activation of H1 receptor and PKC pathway. Histamine may be an endogenous protective factor and calls for its further study as a regulator of astrocyte function during ischemic stroke. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Formyltetrahydrofolate Synthetase Gene Diversity in the Guts of Higher Termites with Different Diets and Lifestyles ▿ †

    Science.gov (United States)

    Ottesen, Elizabeth A.; Leadbetter, Jared R.

    2011-01-01

    In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The “higher” termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the “lower” termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches. PMID:21441328

  3. 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

  4. 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.

  5. Real-time PCR assays targeting formyltetrahydrofolate synthetase gene to enumerate acetogens in natural and engineered environments.

    Science.gov (United States)

    Xu, Kewei; Liu, He; Du, Guocheng; Chen, Jian

    2009-10-01

    Acetogens are ubiquitous in many anaerobic habitats and play a very important role in bioconversion and biodegradation of organic compounds. Methods for rapid detection and quantification of acetogens in different environments are urgently needed to understand the in situ activities in complicated microbial communities. To overcome the limitations of culture-dependent methods and provide enhanced diagnostic tools for determination of the ecological roles of acetogens in different habitats, a quantitative real-time PCR (qrt-PCR) approach targeting functional FTHFS (fhs) gene encoding the formyltetrahydrofolate synthetase was developed. Novel primers flanking the FTHFS fragment were designed and tested. High specificity and sensitivity for estimation of the abundance of acetogens were confirmed analysis of a collection of acetogens, clone libraries and melting curves. The utility of the assay was validated and used in quantifying the FTHFS gene present in different anoxic and oxic habitats, including anoxic and oxic sludges, lake sediment, sewage sullage as well as flooded rice field soils. The abundance of FTHFS gene recovered by fhs1 assay was in the order of magnitude of 10(5) up to 10(7) copies per gram of dry weight sample, and the maximum calculated abundance of acetogens relative to Eubacteria was 0.6-0.9%, confirming the low proportion of acetogens to total bacteria in environments.

  6. Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis

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    Erika M. Palmieri

    2017-08-01

    Full Text Available Glutamine-synthetase (GS, the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.

  7. Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily.

    Science.gov (United States)

    Reuter, K; Mofid, M R; Marahiel, M A; Ficner, R

    1999-12-01

    The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

  8. Localization of two human autoantigen genes by PCR screening and in situ hybridization-glycyl-tRNA synthetase locates to 7p15 and Alanyl-tRNA synthetase locates to 16q22

    Energy Technology Data Exchange (ETDEWEB)

    Nichols, R.C.; Pai, S.I.; Liu, P. [National Inst. of Health, Bethesda, MD (United States); Ge, Q.; Targoff, I.N. [Oklahoma Medical Research Foundation, Oklahoma City, OK (United States)

    1995-11-01

    Aminoacyl-tRNA synthetases (aminoacyl-RS) catalyze the attachment of an amino acid to its cognate tRNA. Five of 20 human aminoacyl-RS (histidyl-RS, threonyl-RS, isoleucyl-RS, glycyl-RS, and alanyl-RS) have been identified as targets of autoantibodies in the autoimmune disease polymyositis/dermatomyositis (PM/DM; 9). A sixth autoantigenic amino-acyl-RS, lysyl-RS, was recently reported. The genes for histidyl-RS and threonyl-RS have been assigned to chromosome 5, as have the genes for leucyl-RS and arginyl-RS. Six other aminoacyl-RS (glutamyl-prolyl-RS, valyl-RS, cysteinyl-RS, methionyl-RS, tryptophanyl-RS, and asparaginyl-RS) were assigned to chromosomes 1, 6, 11, 12, 14, and 18, respectively. The reason for a preponderance of aminoacyl-RS genes on chromosome 5 is unknown, but it has been suggested that regulatory relatedness might be a factor. Recently the entire or partial cDNA sequences for two autoantigenic aminoacyl-RS genes, glycyl-RS (gene symbol GARS; 4) and alanyl-RS (gene symbol AARS; 1), were reported. To understand further the genesis of autoimmune responses to aminoacyl-RS and to determine whether genes for autoantigenic aminoacyl-RS colocalize to chromosome 5, we have determined the chromosomal site of the GARS and AARS genes by PCR-based screening of somatic cell hybrid panels and by fluorescence in situ hybridization (FISH) analysis. 10 refs., 1 fig.

  9. 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

  10. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

    Science.gov (United States)

    Amoutzias, Grigoris D; Chaliotis, Anargyros; Mossialos, Dimitris

    2016-04-16

    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.

  11. 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.

  12. High Throughput Virtual Screening to Identify Novel natural product Inhibitors for MethionyltRNA-Synthetase of Brucella melitensis.

    Science.gov (United States)

    Kumari, Madhulata; Chandra, Subhash; Tiwari, Neeraj; Subbarao, Naidu

    2017-01-01

    The Brucella melitensis methionyl-tRNA-synthetase (MetRSBm) is a promising target for brucellosis drug development. The virtual screening of large libraries of a drug like molecules against a protein target is a common strategy used to identify novel inhibitors. A High throughput virtual screening was performed to identify hits to the potential antibrucellosis drug target, MetRSBm. The best inhibitor identified from the literature survey was 1312, 1415, and 1430. In the virtual screening 56,400 compounds of ChEMBL antimycobacterial library, 1596 approved drugs, 419 Natural product IV library, and 2396 methionine analogous were docked and rescoring, identified top 10 ranked compounds as anti-mycobacterial leads showing G-scores -10.27 to -8.42 (in kcal/mol), approved drugs G-scores -9.08 to -6.60 (in kcal/mol), Natural product IV library G-scores -10.55 to -6.02 (in kcal/mol), methionine analogous Gscores -11.20 to -8.51 (in kcal/mol), and compared with all three known inhibitors (as control) G-scores -3.88 to -3.17 (in kcal/mol). This result indicates these novel compounds have the best binding affinity for MetRSBm. In this study, we extrapolate that the analogous of methionine for find novel drug likeness has been identified [4-(L-histidyl)-2-phenylbenzoyl] methionine hydrochloride, might show the inhibitor of Brucella melitensis effect by interacting with MetRS enzyme. We suggests that Prumycin as a natural product is the novel drugs for brucellosis.

  13. 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/.

  14. 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.

  15. 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.

  16. 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.

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

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    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.

  18. Glutamine synthetase and glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37.

    Science.gov (United States)

    Kainulainen, Veera; Loimaranta, Vuokko; Pekkala, Anna; Edelman, Sanna; Antikainen, Jenni; Kylväjä, Riikka; Laaksonen, Maiju; Laakkonen, Liisa; Finne, Jukka; Korhonen, Timo K

    2012-05-01

    Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

  19. Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase.

    Science.gov (United States)

    Schomer, Rebecca A; Thomas, Michael G

    2017-10-10

    Many nonribosomal peptide synthetases (NRPSs) require MbtH-like proteins (MLPs) for solubility or for activation of amino acid substrate by the adenylation domain. MLPs are capable of functional crosstalk with noncognate NRPSs at varying levels. Using enterobactin biosynthesis in Escherichia coli as a model MLP-dependent NRPS system, we use in vivo and in vitro techniques to characterize how seven noncognate MLPs influence the function of the enterobactin NRPS EntF when the cognate MLP, YbdZ, is absent. Using a series of in vitro assays to analyze EntF solubility, adenylation, aminoacylation, and in vitro enterobactin production, we show that interactions between MLPs and NRPSs are multifaceted and more complex than previously appreciated. We separate MLP influence on solubility and function in a manner that shows altered solubility is not indicative of a functional MLP/NRPS pair. Although much of the functional variation among these noncognates can be explained by differences in EntF affinity for an MLP or the extent an MLP alters EntF l-Ser affinity, we demonstrate that MLPs can have a broader impact beyond solubility and adenylation. First, we show that a noncognate MLP can affect formation of l-Ser-S-EntF. Second, under in vitro conditions saturating for substrate and MLP, enterobactin production remains compromised in the absence of an appropriate MLP partner. These data suggest that we expand our investigations into how the MLPs influence NRPS enzymology. A more detailed understanding of these influences will be essential for downstream engineering of hybrid NRPS systems.

  20. 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.

  1. 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.

  2. 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...... and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots....

  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. Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases in rice.

    Science.gov (United States)

    Yamaya, Tomoyuki; Kusano, Miyako

    2014-10-01

    The functions of the three isoenzymes of cytosolic glutamine synthetase (GS1;1, GS1;2, and GS1;3) and two NADH-glutamate synthases (NADH-GOGAT1 and NADH-GOGAT2) in rice (Oryza sativa L.) were characterized using a reverse genetics approach and spatial expression of the corresponding genes. OsGS1;2 and OsNADH-GOGAT1 were mainly expressed in surface cells of rice roots in an NH4 (+)-dependent manner. Disruption of either gene by the insertion of endogenous retrotransposon Tos17 caused reduction in active tiller number and hence panicle number at harvest. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knockout mutants successfully restored panicle number to wild-type levels. These results indicate that GS1;2 and NADH-GOGAT1 are important in the primary assimilation of NH4 (+) taken up by rice roots. OsGS1;1 and OsNADH-GOGAT2 were mainly expressed in vascular tissues of mature leaf blades. OsGS1;1 mutants showed severe reduction in growth rate and grain filling, whereas OsNADH-GOGAT2 mutants had marked reduction in spikelet number per panicle. Complementation of phenotypes seen in the OsGS1;1 mutant was successfully observed when OsGS1;1 was re-introduced. Thus, these two enzymes could be important in remobilization of nitrogen during natural senescence. Metabolite profiling data showed a crucial role of GS1;1 in coordinating metabolic balance in rice. Expression of OsGS1:3 was spikelet-specific, indicating that it is probably important in grain ripening and/or germination. Thus, these isoenzymes seem to possess distinct and non-overlapping functions and none was able to compensate for the individual function of another. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. 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

  6. 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

  7. Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

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    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

  8. High levels of asparagine synthetase in hypocotyls of pine seedlings suggest a role of the enzyme in re-allocation of seed-stored nitrogen.

    Science.gov (United States)

    Cañas, Rafael A; de la Torre, Fernando; Cánovas, Francisco M; Cantón, Francisco R

    2006-06-01

    A pine asparagine synthetase gene expressed in developing seedlings has been identified by cloning its cDNA (PsAS1) from Scots pine (Pinus sylvestris L.). Genomic DNA analysis with PsAS1 probes and a sequence-based phylogenetic tree are consistent with the possibility of more than one gene encoding asparagine synthetase in pine. However, the parallel patterns of free asparagine content and PsAS1 products indicate that the protein encoded by this gene is mainly responsible for the accumulation of this amino acid during germination and early seedling development. The temporal and spatial patterns of PsAS1 expression together with the spatial distribution of asparagine content suggest that, early after germination, part of the nitrogen mobilized from the megagametophyte is diverted toward the hypocotyl to produce high levels of asparagine as a reservoir of nitrogen to meet later specific demands of development. Furthermore, the transcript and protein analyses in seedlings germinated and growth for extended periods under continuous light or dark suggest that the spatial expression pattern of PsAS1 is largely determined by a developmental program. Therefore, our results suggest that the spatial and temporal control of PsAS1 expression determines the re-allocation of an important amount of seed-stored nitrogen during pine germination.

  9. Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth disease

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Wei; Schimmel, Paul; Yang, Xiang-Lei, E-mail: xlyang@scripps.edu [Departments of Molecular Biology and Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, BCC-379, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2006-12-01

    Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth Disease. Glycyl-tRNA synthetase (GlyRS) is one of a group of enzymes that catalyze the synthesis of aminoacyl-tRNAs for translation. Mutations of human and mouse GlyRSs are causally associated with Charcot–Marie–Tooth disease, the most common genetic disorder of the peripheral nervous system. As the first step towards a structure–function analysis of this disease, native human GlyRS was expressed, purified and crystallized. The crystal belonged to space group P4{sub 3}2{sub 1}2 or its enantiomorphic space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 91.74, c = 247.18 Å, and diffracted X-rays to 3.0 Å resolution. The asymmetric unit contained one GlyRS molecule and had a solvent content of 69%.

  10. Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth disease

    International Nuclear Information System (INIS)

    Xie, Wei; Schimmel, Paul; Yang, Xiang-Lei

    2006-01-01

    Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth Disease. Glycyl-tRNA synthetase (GlyRS) is one of a group of enzymes that catalyze the synthesis of aminoacyl-tRNAs for translation. Mutations of human and mouse GlyRSs are causally associated with Charcot–Marie–Tooth disease, the most common genetic disorder of the peripheral nervous system. As the first step towards a structure–function analysis of this disease, native human GlyRS was expressed, purified and crystallized. The crystal belonged to space group P4 3 2 1 2 or its enantiomorphic space group P4 1 2 1 2, with unit-cell parameters a = b = 91.74, c = 247.18 Å, and diffracted X-rays to 3.0 Å resolution. The asymmetric unit contained one GlyRS molecule and had a solvent content of 69%

  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...

  12. Activity of enzymes that hydrolyze sucrose and raffinose in the first stages of germination of Lactuca sativa cv. Grand rapids. [Invertase, alpha-galactosidose, and sucrose synthetase were observed

    Energy Technology Data Exchange (ETDEWEB)

    Slabnik, E.; Calderon, P.; Diaz, H.

    1981-01-01

    The activities of enzymes capable of metabolizing raffinose and sucrose on achenes of lettuce were studied. During the first stages of germination, evidence was obtained for the occurrence of invertase in the endosperm and embryonic axis. Alpha-galactosidase was localized in the endosperm and cotyledons. Sucrose synthetase was present in the dry seed.

  13. 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

  14. The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection.

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    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.

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

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    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.

  16. NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

    International Nuclear Information System (INIS)

    Kohda, D.; Kawai, G.; Yokoyama, S.; Kawakami, M.; Mizushima, S.; Miyazawa, T.

    1987-01-01

    The 400-MHz 1 H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche + form about the C/sub α/-C/sub β/ bond and the trans form about the C/sub β/-C/sub γ 1 / bond. The IleRS-bound L-valine takes the guache - form about the C/sub α/-C/sub β/ bond. Thus, the conformation of the IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the δ-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases

  17. 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

    International Nuclear Information System (INIS)

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-01-01

    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)

  18. Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145

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    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.

  19. Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase.

    Science.gov (United States)

    Samuel, C E; Rabinowitz, J C

    1974-04-01

    The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA(f) (Met). Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg(2+), and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K(m) values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (l-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA(f) (Met) and tRNA(m) (Met) isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg(2+), and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K(m) value of plus-folate formyltransferase for plus-folate Met-tRNA(f) (Met) does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA(f) (Met). Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases

  20. The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly1

    Science.gov (United States)

    Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Costa, Fabrizio; Aharoni, Asaph

    2015-01-01

    The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning. PMID:26443676

  1. 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 tRNA Leu 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-tRNA Leu and isoleucyl-tRNA Leu . 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.

  2. Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp.

    Science.gov (United States)

    Mofid, Mohammad Reza; Finking, Robert; Marahiel, Mohamed A

    2002-05-10

    The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and

  3. Gene expression in plant lipid metabolism in Arabidopsis seedlings.

    Directory of Open Access Journals (Sweden)

    An-Shan Hsiao

    Full Text Available Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3, DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1 and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6 in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1 and LATE ELONGATED HYPOCOTYL (LHY from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.

  4. Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18.

    Science.gov (United States)

    Bao, Baolong; Pestinger, Valerie; Hassan, Yousef I; Borgstahl, Gloria E O; Kolar, Carol; Zempleni, Janos

    2011-05-01

    Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [(3)H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Acyl coenzyme A synthetase long-chain 1 (ACSL1 gene polymorphism (rs6552828 and elite endurance athletic status: a replication study.

    Directory of Open Access Journals (Sweden)

    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.

  6. The cyclic GMP-AMP synthetase-STING signaling pathway is required for both the innate immune response against HBV and the suppression of HBV assembly.

    Science.gov (United States)

    Dansako, Hiromichi; Ueda, Youki; Okumura, Nobuaki; Satoh, Shinya; Sugiyama, Masaya; Mizokami, Masashi; Ikeda, Masanori; Kato, Nobuyuki

    2016-01-01

    During viral replication, the innate immune response is induced through the recognition of viral replication intermediates by host factor(s). One of these host factors, cyclic GMP-AMP synthetase (cGAS), was recently reported to be involved in the recognition of viral DNA derived from DNA viruses. However, it is uncertain whether cGAS is involved in the recognition of hepatitis B virus (HBV), which is a hepatotropic DNA virus. In the present study, we demonstrated that HBV genome-derived double-stranded DNA induced the innate immune response through cGAS and its adaptor protein, stimulator of interferon genes (STING), in human hepatoma Li23 cells expressing high levels of cGAS. In addition, we demonstrated that HBV infection induced ISG56 through the cGAS-STING signaling pathway. This signaling pathway also showed an antiviral response towards HBV through the suppression of viral assembly. From these results, we conclude that the cGAS-STING signaling pathway is required for not only the innate immune response against HBV but also the suppression of HBV assembly. The cGAS-STING signaling pathway may thus be a novel target for anti-HBV strategies. © 2015 FEBS.

  7. Value and Limits of Routine Histology Alone or Combined with Glutamine Synthetase Immunostaining in the Diagnosis of Hepatocellular Adenoma Subtypes on Surgical Specimens

    Directory of Open Access Journals (Sweden)

    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.

  8. Diversity of formyltetrahydrofolate synthetase genes in the rumens of roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed different diets.

    Science.gov (United States)

    Li, Zhipeng; Henderson, Gemma; Yang, Yahan; Li, Guangyu

    2017-01-01

    Reductive acetogenesis by homoacetogens represents an alternative pathway to methanogenesis to remove metabolic hydrogen during rumen fermentation. In this study, we investigated the occurrence of homoacetogen in the rumens of pasture-fed roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed either oak-leaf-based (tannin-rich, 100 mg/kg dried matter), corn-stover-based, or corn-silage-based diets, by using formyltetrahydrofolate synthetase (FTHFS) gene sequences as a marker. The diversity and richness of FTHFS sequences was lowest in animals fed oak leaf, indicating that tannin-containing plants may affect rumen homoacetogen diversity. FTHFS amino acid sequences in the rumen of roe deer significantly differed from those of sika deer. The phylogenetic analyses showed that 44.8% of sequences in pasture-fed roe deer, and 72.1%, 81.1%, and 37.5% of sequences in sika deer fed oak-leaf-, corn-stover-, and corn-silage-based diets, respectively, may represent novel bacteria that have not yet been cultured. These results demonstrate that the rumens of roe deer and sika deer harbor potentially novel homoacetogens and that diet may influence homoacetogen community structure.

  9. 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.

  10. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte–neuron co-cultures

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-01-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of 15NH4+ in alanine during acute hyperammonemia. We observed a fourfold increased amount of 15NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to 15NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of 15NH4 into alanine together with increased 15N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy. PMID:23673435

  11. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures.

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-08-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH4(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH4 into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

  12. IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

    Directory of Open Access Journals (Sweden)

    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.

  13. Concurrent overactivation of the cytosolic glutamine synthetase and the GABA shunt in the ABA-deficient sitiens mutant of tomato leads to resistance against Botrytis cinerea.

    Science.gov (United States)

    Seifi, Hamed Soren; Curvers, Katrien; De Vleesschauwer, David; Delaere, Ilse; Aziz, Aziz; Höfte, Monica

    2013-07-01

    Deficiency of abscisic acid (ABA) in the sitiens mutant of tomato (Solanum lycopersicum) culminates in increased resistance to Botrytis cinerea through a rapid epidermal hypersensitive response (HR) and associated phenylpropanoid pathway-derived cell wall fortifications. This study focused on understanding the role of primary carbon : nitrogen (C : N) metabolism in the resistance response of sitiens to B. cinerea. How alterations in C : N metabolism are linked with the HR-mediated epidermal arrest of the pathogen has been also investigated. Temporal alterations in the γ-aminobutyric acid (GABA) shunt, glutamine synthetase/glutamate synthase (GS/GOGAT) cycle and phenylpropanoid pathway were transcriptionally, enzymatically and metabolically monitored in both wild-type and sitiens plants. Virus-induced gene silencing, microscopic analyses and pharmacological assays were used to further confirm the data. Our results on the sitiens-B. cinerea interaction favor a model in which cell viability in the cells surrounding the invaded tissue is maintained by a constant replenishment of the tricarboxylic acid (TCA) cycle through overactivation of the GS/GOGAT cycle and the GABA shunt, resulting in resistance through both tightly controlling the defense-associated HR and slowing down the pathogen-induced senescence. Collectively, this study shows that maintaining cell viability via alterations in host C : N metabolism plays a vital role in the resistance response against necrotrophic pathogens. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  14. 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

  15. Mutations in LACS2, a long-chain acyl-coenzyme A synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to Botrytis cinerea in Arabidopsis.

    Science.gov (United States)

    Tang, Dingzhong; Simonich, Michael T; Innes, Roger W

    2007-06-01

    We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea.

  16. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

    Carbon and nitrogen are the most abundant elements in plants, together making up around 40-50% and 2-6% of dry matter respectively. Elevated atmospheric CO2 levels are predicted to double by the end of this century, increasing carbon fixation by C3 plants like Arabidopsis and, hence, their carbon...... of the complicated interactions between nitrogen and carbon, pointing towards the need for a deeper understanding of the same.......Carbon and nitrogen are the most abundant elements in plants, together making up around 40-50% and 2-6% of dry matter respectively. Elevated atmospheric CO2 levels are predicted to double by the end of this century, increasing carbon fixation by C3 plants like Arabidopsis and, hence, their carbon...... 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...

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions.

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    Sharma, Richa; Jamwal, Vijaylakshmi; Singh, Varun P; Wazir, Priya; Awasthi, Praveen; Singh, Deepika; Vishwakarma, Ram A; Gandhi, Sumit G; Chaubey, Asha

    2017-07-10

    Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K + ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC-MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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.

  3. Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens.

    Science.gov (United States)

    Fang, Haitian; Liu, Huiyan; Chen, Ning; Zhang, Chenglin; Xie, Xixian; Xu, Qingyang

    2013-06-01

    A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

  4. Detection of active, potentially acetate-oxidizing syntrophs in an anaerobic digester by flux measurement and formyltetrahydrofolate synthetase (FTHFS) expression profiling.

    Science.gov (United States)

    Hori, Tomoyuki; Sasaki, Daisuke; Haruta, Shin; Shigematsu, Toru; Ueno, Yoshiyuki; Ishii, Masaharu; Igarashi, Yasuo

    2011-07-01

    Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial approach of flux measurement and transcriptional profiling of the formyltetrahydrofolate synthetase (FTHFS) gene, an ecological biomarker for reductive acetogenesis. During the operation of a thermophilic anaerobic digester, volatile fatty acids were mostly depleted, suggesting a high turnover rate for dissolved H(2), and hydrogenotrophic methanogens were the dominant archaeal members. Batch cultivation of the digester microbiota with (13)C-labelled acetate indicated that syntrophic oxidation accounted for 13.1-21.3 % of methane production from acetate. FTHFS genes were transcribed in the absence of carbon monoxide, methoxylated compounds and inorganic electron acceptors other than CO(2), which is implicated in the activity of reversed reductive acetogenesis; however, expression itself does not distinguish whether biosynthesis or biodegradation is functioning. The mRNA- and DNA-based terminal RFLP and clone library analyses indicated that, out of nine FTHFS phylotypes detected, the FTHFS genes from the novel phylotypes I-IV in addition to the known syntroph Thermacetogenium phaeum (i.e. phylotype V) were specifically expressed. These transcripts arose from phylogenetically presumed homoacetogens. The results of this study demonstrate that hitherto unidentified phylotypes of homoacetogens are responsible for syntrophic acetate oxidation in an anaerobic digester.

  5. 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.

  6. 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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants.

    Science.gov (United States)

    Spena, A; Prinsen, E; Fladung, M; Schulze, S C; Van Onckelen, H

    1991-06-01

    The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[14C]lysine when fed with [14C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterized by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.

  9. A novel mutation of the glycyl-tRNA synthetase (GARS) gene associated with Charcot-Marie-Tooth type 2D in a Chinese family.

    Science.gov (United States)

    Sun, Aping; Liu, Xiangyi; Zheng, Mei; Sun, Qingli; Huang, Yu; Fan, Dongsheng

    2015-09-01

    To explore the clinical features of a novel glycyl-tRNA synthetase (GARS) gene mutation in a family with Charcot-Marie-Tooth disease type 2D (CMT2D). Exome capture with the next-generation sequencing technique was used to detect gene mutations. The mutations were verified by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique combined with DNA sequencing. In this pedigree, eight members were affected; seven males and one female. The affected members initially manifested with the onset of hand muscle weakness and atrophy in adolescence followed by gradual development of distal lower limb involvement and minor sensory involvement. Electrophysiological studies revealed that this disease mainly involves axonal damage. Genetic detection showed that all affected family members had a heterozygous missense mutation, c.999G>T (p.E333D), of the GARS gene. The c.999G>T mutation is a novel mutation of the GARS gene that has not been previously reported. The phenotype of this family is CMT2D, which is first reported in Chinese population.

  10. Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress

    Science.gov (United States)

    Ma, Chunquan; Wang, Yuguang; Gu, Dan; Nan, Jingdong; Chen, Sixue; Li, Haiying

    2017-01-01

    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. PMID:28420190

  11. Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.

    Science.gov (United States)

    Thorgersen, Michael P; Lipscomb, Gina L; Schut, Gerrit J; Kelly, Robert M; Adams, Michael W W

    2014-03-01

    The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus. Copyright © 2014 International Metabolic Engineering Society. All rights reserved.

  12. Engineering of recombinant Escherichia coli cells co-expressing poly-γ-glutamic acid (γ-PGA) synthetase and glutamate racemase for differential yielding of γ-PGA.

    Science.gov (United States)

    Cao, Mingfeng; Geng, Weitao; Zhang, Wei; Sun, Jibin; Wang, Shufang; Feng, Jun; Zheng, Ping; Jiang, Anna; Song, Cunjiang

    2013-11-01

    Poly-γ-glutamic acid (γ-PGA) is a promising environmental-friendly material with outstanding water solubility, biocompatibility and degradability. However, it is tough to determine the relationship between functional synthetic enzyme and the strains' yield or substrate dependency. We cloned γ-PGA synthetase genes pgsBCA and glutamate racemase gene racE from both L-glutamate-dependent γ-PGA-producing Bacillus licheniformis NK-03 and L-glutamate-independent B. amyloliquefaciens LL3 strains. The deduced RacE and PgsA from the two strains shared the identity of 84.5% and 78.53%, while PgsB and PgsC possessed greater similarity with 93.13% and 93.96%. The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing γ-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria-Bertani medium containing glucose or L-glutamate. However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon γ-PGA production. Furthermore, RacE integration could enhance γ-PGA yield through improving the preferred d-glutamate content. This is the first report about co-expression of pgsBCA and racE from the two Bacillus strains, which will be of great value for the determination of the biosynthetic mechanism of γ-PGA. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation.

    Science.gov (United States)

    Wu, Xiaobin; García-Estrada, Carlos; Vaca, Inmaculada; Martín, Juan-Francisco

    2012-02-01

    The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs (3371)EGHGRE(3376) (located in the putative epimerase domain) and (3629)GWSFG(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thioesterease interacts with the other ACVS domains. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  14. Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides.

    Science.gov (United States)

    Schauwecker, F; Pfennig, F; Grammel, N; Keller, U

    2000-04-01

    Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

  15. 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.

  16. 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.

  17. Arabidopsis acetyl-amido synthetase GH3.5 involvement in camalexin biosynthesis through conjugation of indole-3-carboxylic acid and cysteine and upregulation of camalexin biosynthesis genes.

    Science.gov (United States)

    Wang, Mu-Yang; Liu, Xue-Ting; Chen, Ying; Xu, Xiao-Jing; Yu, Biao; Zhang, Shu-Qun; Li, Qun; He, Zu-Hua

    2012-07-01

    Camalexin (3-thiazol-2'-yl-indole) is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile (IAN) is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Arabidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid (DHCA), the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1D/pad3-1, suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys), and upregulation of the major biosynthetic pathway genes. © 2012 Institute of Botany, Chinese Academy of Sciences.

  18. 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.

  19. 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

  20. 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.

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    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.

  1. A sub-threshold dose of pilocarpine increases glutamine synthetase in reactive astrocytes and enhances the progression of amygdaloid-kindling epilepsy in rats.

    Science.gov (United States)

    Sun, Hong-Liu; Deng, Da-Ping; Pan, Xiao-Hong; Wang, Chao-Yun; Zhang, Xiu-Li; Chen, Xiang-Ming; Wang, Chun-Hua; Liu, Yu-Xia; Li, Shu-Cui; Bai, Xian-Yong; Zhu, Wei

    2016-03-02

    The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

  2. 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 < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. 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.

  3. A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat.

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    Hong-Liu Sun

    Full Text Available Reduction of glutamine synthetase (GS function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl, a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated. It was found that low doses of MSO (5 or 10 µg significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×10(6 TU/2 µl of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.

  4. Long chain fatty Acyl-CoA synthetase 4 is a biomarker for and mediator of hormone resistance in human breast cancer.

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    Xinyu Wu

    Full Text Available The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4 in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2 in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC, characterized by the absence of androgen receptor (AR and therefore referred to as quadruple negative breast cancer (QNBC. Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.

  5. Potential of Krebs von den Lungen-6 as a predictor of relapse in interstitial pneumonia with anti-aminoacyl tRNA synthetase antibodies-positive dermatomyositis.

    Science.gov (United States)

    Isoda, Kentaro; Kotani, Takuya; Takeuchi, Tohru; Konma, Junichi; Ishida, Takaaki; Hata, Kenichiro; Otani, Kenichiro; Fujiwara, Hiroshi; Shoda, Takeshi; Makino, Shigeki; Arawaka, Shigeki

    2018-04-06

    To identify a predictor of relapse in interstitial pneumonia (IP) in patients with anti-aminoacyl tRNA synthetase antibodies-positive dermatomyositis (ARS-DMIP). This retrospective cohort study comprised 27 ARS-DMIP patients. We compared clinical and laboratory findings between the relapse and non-relapse groups during 2 years after treatment initiation to find predictors of relapse in IP. Candidate predictors were further assessed by analysing the relationship with the relapse of IP. One patient with ARS-DMIP died. 7 (26.9%) of the remaining 26 patients with ARS-DMIP had a relapse of IP. We found that the levels of serum Krebs von den Lungen-6 (KL-6) in the relapse group were significantly higher than those in the non-relapse group at the time points before treatment (P = 0.046) and after treatments, including 6 (P = 0.004), 12 (P =0013), 18 (P =0.003) and 24 months (P <0.001). The KL-6 values that maximised the area under the ROC curve were 2347 U/mL before treatment, 622 U/mL after 6 months and 468 U/mL after 12 months. The relapse rates after 104 weeks were significantly higher in patients with KL-6 levels ≥2400 U/mL before treatment (P = 0.014), ≥600 ng/mL after 6 months (P < 0.005), and ≥470 U/mL after 12 months (P = 0.010). These findings suggest that the levels of KL-6 before and after treatment in ARS-DMIP may represent the disease activity of IP, and they may be useful as the predictor of relapse in IP in patients with ARS-DMIP. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

  6. Stable Analogues of OSB-AMP: Potent Inhibitors of MenE the o-succinylbenzoate-CoA Synthetase from Bacterial Menaquinone Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lu X.; Swaminathan S.; Zhou R.; Sharma I.; Li X.; Kumar G.; Tonge P. J.; Tan D. S.

    2012-01-02

    MenE, the o-succinylbenzoate (OSB)-CoA synthetase from bacterial menaquinone biosynthesis, is a promising new antibacterial target. Sulfonyladenosine analogues of the cognate reaction intermediate, OSB-AMP, have been developed as inhibitors of the MenE enzymes from Mycobacterium tuberculosis (mtMenE), Staphylococcus aureus (saMenE) and Escherichia coli (ecMenE). Both a free carboxylate and a ketone moiety on the OSB side chain are required for potent inhibitory activity. OSB-AMS (4) is a competitive inhibitor of mtMenE with respect to ATP (K{sub i} = 5.4 {+-} 0.1 nM) and a noncompetitive inhibitor with respect to OSB (K{sub i} = 11.2 {+-} 0.9 nM). These data are consistent with a Bi Uni Uni Bi Ping-Pong kinetic mechanism for these enzymes. In addition, OSB-AMS inhibits saMenE with K{sub i}{sup app} = 22 {+-} 8 nM and ecMenE with K{sub i}{sup OSB} = 128 {+-} 5 nM. Putative active-site residues, Arg222, which may interact with the OSB aromatic carboxylate, and Ser302, which may bind the OSB ketone oxygen, have been identified through computational docking of OSB-AMP with the unliganded crystal structure of saMenE. A pH-dependent interconversion of the free keto acid and lactol forms of the inhibitors is also described, along with implications for inhibitor design.

  7. Genetic polymorphisms of the serotonin transporter, but not the 2a receptor or nitric oxide synthetase, are associated with pulmonary hypertension in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Ulrich, Silvia; Hersberger, Martin; Fischler, Manuel; Nussbaumer-Ochsner, Yvonne; Treder, Ursula; Russi, Erich W; Speich, Rudolf

    2010-01-01

    Pulmonary hypertension (PH) is prognosti- cally important in chronic obstructive pulmonary disease (COPD). Since PH only weakly correlates with hypoxemia, other factors must play a role. To investigate whether polymorphisms of the serotonin transporter (5HTT), serotonin-2a receptor (5HTR2a) and endothelial nitric oxide synthetase (eNOS) are related to PH in COPD. In 59 COPD patients who underwent right heart catheterization, 6-min walking distance, NYHA functional class, pulmonary function tests, blood gases and 5HTT, 5HTR2a and eNOS (4ab and T298C) polymorphisms were determined. Forty-nine COPD patients in NYHA functional class III-IV were included. Ten were excluded due to comorbid causes of PH (mainly chronic thromboembolic). PH (mPAP > or =25 mm Hg) was present in 55% and usually mild, but out of proportion (mPAP > or =40 mm Hg) in 12%. Patients with PH had significantly higher frequencies of the 5HTT-L-allele (52%) compared to individuals without PH (36%), and LL homozygote patients had more severe PH. In patients with out-of-proportion PH, the L-allelic frequency was even 75%. We found no association of 5HTR2a and eNOS polymorphism with PH in COPD. In this COPD cohort we confirm that PH is frequent and usually mild, but out of proportion in a subgroup. We found a significant association of the L-allelic variant of 5HTT with PH overall and especially in out-of-proportion PH. These findings may point towards a role of the serotonin system in COPD-PH and warrant further studies. Copyright 2009 S. Karger AG, Basel.

  8. Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

    Science.gov (United States)

    Galloway, D C; Blake, D G; Shepherd, A G; McLellan, L I

    1997-11-15

    We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

  9. Conformation change of tRNA/sub Glu/ in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

    International Nuclear Information System (INIS)

    Hara-Yokoyama, M.; Yokoyama, S.; Miyazawa, T.

    1986-01-01

    The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNA/sup Glu/, Escherichia coli tRNA/sup Glu/, and amino acids was studied by fluorescence measurements. In the absence of tRNA/sup Glu/, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E.coli tRNA/sup Glu/, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (K/sub m/) for tRNA/sup Glu/, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNA/sup Glu/ and heterologous tRNA/sup Glu/ species. As the KCl concentration is raised from 0 to 100 mM, the K/sub m/ value for L-glutamate in the heterologous system is remarkably increased whereas the K/sub m/ value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNA/sup Glu/ in the complex. The conformation change of T. thermophilus tRNA/sup Glu/ upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNA/sup Glu/GluRS complex is in equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small K/sub m/ value for L-glutamate. In this form of the complex, the conformation of tRNA/sup Glu/ is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNA/sup Glu/ in the complex with GluRS is required for the specific binding of L-glutamate as the substrate

  10. Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings.

    Science.gov (United States)

    Ohashi, Miwa; Ishiyama, Keiki; Kusano, Miyako; Fukushima, Atsushi; Kojima, Soichi; Hanada, Atsushi; Kanno, Keiichi; Hayakawa, Toshihiko; Seto, Yoshiya; Kyozuka, Junko; Yamaguchi, Shinjiro; Yamaya, Tomoyuki

    2015-01-01

    The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the phloem companion cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  11. 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.

  12. 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.

  13. γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

    Science.gov (United States)

    Lin, Li-Ching; Chen, Chi-Fen; Ho, Chun-Te; Liu, Jun-Jen; Liu, Tsan-Zon; Chern, Chi-Liang

    2018-04-01

    This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ‑glutamylcysteine synthetase heavy chain (γ‑GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ‑GCSh activity were measured by HPLC methods. γ‑GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ‑GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ‑GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. Our data unequivocally demonstrate that γ‑GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Mutant glycyl-tRNA synthetase (Gars ameliorates SOD1(G93A motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice.

    Directory of Open Access Journals (Sweden)

    Gareth T Banks

    2009-07-01

    Full Text Available In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs.We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous Gars(C201R/+ mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the Gars(C201R/+ mice to two other mutants: the TgSOD1(G93A model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1(Loa which has a defect in the heavy chain of the dynein complex. We found the Dync1h1(Loa/+;Gars(C201R/+ double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the Gars(C201R mutation significantly delayed disease onset in the SOD1(G93A;Gars(C201R/+ double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated.These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

  15. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis....

  16. 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

    CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization...... and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition...

  17. Variation in Antiviral 2', 5'-Oligoadenylate Synthetase (2'5'AS) Enzyme Activity is controlled by a Single-Nucleotide Polymorphism at a Splice-Acceptor Site in the OAS1 Gene

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V.; Leigh, F.L.; Lu, S.

    2005-01-01

    It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine......, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1...

  18. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana[C][W][OA

    Science.gov (United States)

    Kim, Sung Soo; Grienenberger, Etienne; Lallemand, Benjamin; Colpitts, Che C.; Kim, Sun Young; Souza, Clarice de Azevedo; Geoffroy, Pierrette; Heintz, Dimitri; Krahn, Daniel; Kaiser, Markus; Kombrink, Erich; Heitz, Thierry; Suh, Dae-Yeon; Legrand, Michel; Douglas, Carl J.

    2010-01-01

    Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors. PMID:21193570

  19. 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

  20. 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

  1. 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

  2. Clinical follow-up predictors of disease pattern change in anti-Jo1 positive anti-synthetase syndrome: Results from a multicenter, international and retrospective study.

    Science.gov (United States)

    Bartoloni, Elena; Gonzalez-Gay, Miguel A; Scirè, Carlo; Castaneda, Santos; Gerli, Roberto; Lopez-Longo, Francisco Javier; Martinez-Barrio, Julia; Govoni, Marcello; Furini, Federica; Pina, Trinitario; Iannone, Florenzo; Giannini, Margherita; Nuño, Laura; Quartuccio, Luca; Ortego-Centeno, Norberto; Alunno, Alessia; Specker, Christopher; Montecucco, Carlomaurizio; Triantafyllias, Konstantinos; Balduzzi, Silvia; Sifuentes-Giraldo, Walter Alberto; Paolazzi, Giuseppe; Bravi, Elena; Schwarting, Andreas; Pellerito, Raffaele; Russo, Alessandra; Selmi, Carlo; Saketkoo, Lesley-Ann; Fusaro, Enrico; Parisi, Simone; Pipitone, Nicolò; Franceschini, Franco; Cavazzana, Ilaria; Neri, Rossella; Barsotti, Simone; Codullo, Veronica; Cavagna, Lorenzo

    2017-03-01

    Arthritis, myositis and interstitial lung disease (ILD) constitute the classic clinical triad of anti-synthetase syndrome (ASSD). These patients experience other accompanying features, such as Raynaud's phenomenon, fever or mechanic's hands. Most ASSD patients develop the complete triad during the follow-up. In the present study we aimed to determine whether the subsequent appearance of accompanying features may suggest the development of triad findings lacking at the onset in anti-Jo1 positive ASSD patients. Anti-Jo1 positive patients presenting with incomplete ASSD (no >2 classic triad features) were assessed. Clinical characteristics and clusters of disease manifestations were retrospectively collected and analyzed in a large international multicenter cohort of ASSD patients. 165 patients (123 women) with incomplete ASSD were identified. Ninety-five patients (57.5%) developed new classic triad manifestations after 15months median (IQR 9-51) and 40 (24%) developed new accompanying features after 19months median (IQR 6-56) from disease onset. During the follow-up, the ex-novo occurrence of triad features was observed in 32 out of 40 patients (80%) with new accompanying findings and in 63 out of 125 patients (50.5%) without new accompanying findings (p=0.002). In patients with at least one new accompanying feature the odds ratio for the occurrence of new triad manifestations was 3.94 with respect to patients not developing ex-novo accompanying findings (95% CI 1.68-9.21, p=0.002). Anti-Jo1 ASSD patients with incomplete forms at disease onset are at high risk for the subsequent occurrence of lacking classic triad findings. Although all ASSD patients should be carefully assessed for the occurrence of new triad features, a closer follow-up should be considered in the subgroup of patients developing ex novo accompanying findings. These patients, indeed, have near four-fold increased risk for new classic triad manifestation occurrence with respect to patients not

  3. 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.

  4. 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

  5. Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Cura, Vincent; Olieric, Natacha; Guichard, Alexandre [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Wang, En-Duo [State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031 (China); Moras, Dino [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Eriani, Gilbert [Architecture et Réactivité de l’ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 Rue René Descartes, 67084 Strasbourg (France); Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

    2005-10-01

    The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques.

  6. Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

    International Nuclear Information System (INIS)

    Cura, Vincent; Olieric, Natacha; Guichard, Alexandre; Wang, En-Duo; Moras, Dino; Eriani, Gilbert; Cavarelli, Jean

    2005-01-01

    The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques

  7. A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

    Directory of Open Access Journals (Sweden)

    Jan Mareš

    Full Text Available A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea. Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

  8. Non-ribosomal peptide synthetases

    Indian Academy of Sciences (India)

    2017-01-19

    Jan 19, 2017 ... 2009). In other words, the characterization of A-domains can help in the structure prediction of the NRP, encoded by a cryptic NRPS BGC. A-domain specificity can also be altered for the synthesis of unnatural products with novel physicochemical properties over the original natural product (Hur et al. 2012).

  9. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    GS activity in root and stem during the vegetative growth stages and an increased GS activity in leaves during senescence compared to wildtype control. Furthermore, during the vegetative growth stages, there were distinct differences in N accumulation and biomass partitioning between transgenic lines...... new insight into the adaptability of plants to fluctuations in N supply. There was a clear relationship between nitrate high affinity uptake and the transcript levels of high affinity transporter, HvNRT2s, suggesting that these may play a dynamic and important role in fine-tuning plant nitrate supply...... for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root...

  10. Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7T during Degradation of 3,3′-Dithiodipropionic Acid ▿ †

    Science.gov (United States)

    Schürmann, Marc; Wübbeler, Jan Hendrik; Grote, Jessica; Steinbüchel, Alexander

    2011-01-01

    The sucCD gene of Advenella mimigardefordensis strain DPN7T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCDAm) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCDAm is Mg2+ or Mn2+ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (Vmax = 9.85 ± 0.14 μmol min−1 mg−1, Km = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (Vmax = 0.12 ± 0.01 μmol min−1 mg−1) and the affinity was 6-fold lower (Km = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCDAm is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP. PMID:21515777

  11. Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation.

    Science.gov (United States)

    Rosowsky, A; Forsch, R A; Reich, V E; Freisheim, J H; Moran, R G

    1992-05-01

    5-Deazafolate and 5-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic acid (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Orn) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of interest as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of N2-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by N10-formylation, mixed anhydride condensation of the resultant N2-acetyl-N10-formyl-5- deazapteroic acid with L-homocysteic acid, and removal of the N2-acetyl and N10-formyl groups with NaOH, afforded N-(5-deazapteroyl)-L-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of N2-acetyl-N10-formyl- 5-deazapteroic acid with methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid, followed by consecutive treatment with Me3SiBr and NaOH, yielded D,L-2-[(5-deazapteroyl)amino]-4-phosphonobutanoic acid (5-dPteAPBA). Treatment with NaOH alone led to retention of one ethyl ester group on the phosphonate moiety. Catalytic hydrogenation of N2-acetyl-N10-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocysteate and deprotection with NaOH afforded N-(5,6,7,8-tetrahydro-5-deazapteroyl)-L-homocysteic acid (5-dH4PteHCysA). Similar chemistry starting from methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl N delta-(benzyloxycarbonyl)-L-ornithinate yielded D,L-2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobut ano ic acid (5-dH4Pte-APBA) and N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine (5-dH4PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT

  12. The aminoacyl-tRNA synthetases had only a marginal role in the origin of the organization of the genetic code: Evidence in favor of the coevolution theory.

    Science.gov (United States)

    Di Giulio, Massimo

    2017-11-07

    The coevolution theory of the origin of the genetic code suggests that the organization of the genetic code coevolved with the biosynthetic relationships between amino acids. The mechanism that allowed this coevolution was based on tRNA-like molecules on which-this theory-would postulate the biosynthetic transformations between amino acids to have occurred. This mechanism makes a prediction on how the role conducted by the aminoacyl-tRNA synthetases (ARSs), in the origin of the genetic code, should have been. Indeed, if the biosynthetic transformations between amino acids occurred on tRNA-like molecules, then there was no need to link amino acids to these molecules because amino acids were already charged on tRNA-like molecules, as the coevolution theory suggests. In spite of the fact that ARSs make the genetic code responsible for the first interaction between a component of nucleic acids and that of proteins, for the coevolution theory the role of ARSs should have been entirely marginal in the genetic code origin. Therefore, I have conducted a further analysis of the distribution of the two classes of ARSs and of their subclasses-in the genetic code table-in order to perform a falsification test of the coevolution theory. Indeed, in the case in which the distribution of ARSs within the genetic code would have been highly significant, then the coevolution theory would be falsified since the mechanism on which it is based would not predict a fundamental role of ARSs in the origin of the genetic code. I found that the statistical significance of the distribution of the two classes of ARSs in the table of the genetic code is low or marginal, whereas that of the subclasses of ARSs statistically significant. However, this is in perfect agreement with the postulates of the coevolution theory. Indeed, the only case of statistical significance-regarding the classes of ARSs-is appreciable for the CAG code, whereas for its complement-the UNN/NUN code-only a marginal

  13. Inhibition of gamma-ray dose-rate effects by D/sup 2/O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, A.M.; Tanaka, O.; Matsudaira, H.

    1984-06-01

    Effects of deuterium oxide (D/sub 2/O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of ..gamma.. rays. Growth of irradiated and unirradiated cells was inhibited by 45% D/sub 2/O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of ..gamma.. rays. Possible mechanisms underlying the inhibition are discussed.

  14. Synthesis research of squalene synthetase inhibitor CP-263, 114. How is skeleton construction carried out?; Sukuaren gosei koso sogaizai CP-263,114 no gosei kenkyu - ikanishite kokkaku kochiku wo okonauka?

    Energy Technology Data Exchange (ETDEWEB)

    Matsushima, Y. [Tokyo Inst. of Tech., Tokyo (Japan)

    2000-01-01

    CP-263, 114 isolated as a squalene synthetase inhibitor and the decyclization of CP-225, 917 was not only expected to be a lead chemical compound of the hypercholesterolemia medicine, but also have collected the attention of the organic synthetic chemistry researchers all over the world from the ring structure of advanced oxygen functionalization. In the CP- chemical compound, the constructive method of the bicyclo ring structure is a key of the synthesis, there are three reports to use the intramolecular Diels-Alder reaction (Fukuyama) and the siloxy-Cope rearrangement (Leighton), intramolecular Heck reaction (Danishefsky) as a result of succeeding in including the foothold to the side-chain lactol ring. Recently, Nicolaou et al. succeeded for the first time in the total synthesis racemic modification shell. They carried out the skeleton construction by the intramolecular Diels-Alder reaction, and constructed the maleic anhydride structure in taking the ketone as a foothold. (NEDO)

  15. Prediction of Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in vivo by mutations in the dihydrofolate reductase and dihydropteroate synthetase genes: a comparative study between sites of differing endemicity

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonja; Khalil, Insaf F

    2003-01-01

    in vivo. The prevalence of mutations in dhfr and dhps in relation to S/P efficacy was studied in four sites of differing endemicity in Sudan, Mozambique, and Tanzania. The sites were organized in order of increasing resistance and a significant increase in the prevalence of triple mutations in codons c51...... recently. However, changes in susceptibility within the same area with moderate levels of resistance may be possible by longitudinal surveillance of a subset of dhfr/dhps mutations that has been associated with S/P resistance in vivo in a defined location.......Plasmodium falciparum resistance to sulfadoxine/pyrimethamine (S/P) is due to mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhfr) genes. Large-scale screening of the prevalence of these mutations could facilitate the surveillance of the level of S/P resistance...

  16. The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly.

    Science.gov (United States)

    Lashbrooke, Justin; Adato, Avital; Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Granell, Antonio; Costa, Fabrizio; Aharoni, Asaph

    2015-12-01

    The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  18. tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP: mechanistic and evolutionary implications.

    Science.gov (United States)

    Blais, Sébastien P; Kornblatt, Jack A; Barbeau, Xavier; Bonnaure, Guillaume; Lagüe, Patrick; Chênevert, Robert; Lapointe, Jacques

    2015-01-01

    For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of -TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3'-OH oxygen of the 3'-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

  19. N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize Partição de nitrogênio e atividade das enzimas nitrato redutase e glutamina sintetase em duas cultivares contrastantes de milho

    Directory of Open Access Journals (Sweden)

    Altair Toledo Machado

    2001-02-01

    Full Text Available In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient and Catetão (inefficient were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction and nitrate reductase did not differ among the varieties.Com o objetivo de identificar parâmetros que possam ser utilizados em programas de melhoramento genético em milho para uso eficiente de N, duas cultivares de milho contrastantes quanto ao uso deste nutriente, Sol da Manhã NF (eficiente e Catetão (não-eficiente, foram avaliadas em dois experimentos conduzidos no campo e em casa de vegetação, respectivamente, sob nível baixo e alto de N. Os caracteres avaliados foram: teor e conteúdo de N em diferentes partes da planta; massa seca; peso dos grãos e de diferentes partes da planta; biomassa, e atividade das enzimas nitrato redutase e glutamina sintetase. O mecanismo de translocação de N e de fotoassimilados para os grãos e a relação fonte/dreno foram importantes para diferenciar a cultivar eficiente da não-eficiente. Conteúdo de N nos grãos e total das plantas, peso do sabugo e relação peso de espiga/matéria seca foram importantes para caracterizar a cultivar Sol da Manhã NF eficiente no uso do N. A

  20. The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

    LENUS (Irish Health Repository)

    Foy, Niall

    2010-07-22

    Abstract Background Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. Results A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. Conclusions The T box element controlling lysK (encoding LysRS1) expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.

  1. [Effects of TCM treatment according to syndrome differentiation on expressions of nuclear factor-kappaB and gamma-glutamylcysteine synthetase in rats with chronic obstructive pulmonary disease of various syndrome types].

    Science.gov (United States)

    Zhang, Wei; Zhang, Xin-Yue; Shao, Yu-Meng

    2007-05-01

    To explore the mechanism of traditional Chinese medicine (TCM) treatment according to syndrome differentiation in treating chronic obstructive pulmonary disease (COPD) by observing the changes of nuclear factor-kappaB (NF-kappaB) and gamma-glutamylcysteine synthetase (gamma-GCS) expression levels in rats. COPD model was established by modified method of combining fumigation and lipopolysaccharide (LPS) intra-tracheal dripping. Model rats were treated respectively for succesive 14 days according to their syndrome, that is, Xiaoqinglong Decoction to the rats of cold-phlegm accumulation in Fei, Maxing Shigan Decoction to those of heat-phlegm accumulation in Fei, Yupingfeng Decoction to those of Fei-qi deficiency, Liujunzi Decoction to those of Pi-qi deficiency, Renshen Gejie Decoction to those of Shen qi-deficiency. Besides, model rats in the model control group received 2mL normal saline daily, and no intervention was applied in the normal control group. The expression of gamma-GCS and NF-kappaB was detected by immunochemistry before and after treatment. Compared with that in the normal rats, the expressions of gamma-GCS and NF-kappaB in bronchial and alveolar epithelium of COPD rats before treatment were significantly higher, but the positive expression rates were lowered after treatment significantly (Psyndrome differentiation could rectify imbalance of oxidation/anti-oxidation and alleviate inflammatory reaction in COPD rats, thus to treat COPD effectively.

  2. Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers

    International Nuclear Information System (INIS)

    Dessen, P.; Ducruix, A.; May, R.P.; Blanquet, S.

    1990-01-01

    Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A)

  3. A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

    International Nuclear Information System (INIS)

    Hickey, E.W.

    1988-01-01

    Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH 0 ) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH 0 induced behavior in E. coli and S. typhimurium by quantitation of H 2 35 SO 4 labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH 0 5.0 or shifted from growth at pH 0 7.0 to 5.0. Internal pH (pH i ) changes occurring during steady state growth at low pH 0 , and on shifting from pH 0 7.0 to 5.0, were measured using 14 C-benzoic acid uptake

  4. Accumulated Expression Level of Cytosolic Glutamine Synthetase 1 Gene (OsGS1;1 or OsGS1;2) Alter Plant Development and the Carbon-Nitrogen Metabolic Status in Rice

    Science.gov (United States)

    Bao, Aili; Zhao, Zhuqing; Ding, Guangda; Shi, Lei; Xu, Fangsen; Cai, Hongmei

    2014-01-01

    Maintaining an appropriate balance of carbon to nitrogen metabolism is essential for rice growth and yield. Glutamine synthetase is a key enzyme for ammonium assimilation. In this study, we systematically analyzed the growth phenotype, carbon-nitrogen metabolic status and gene expression profiles in GS1;1-, GS1;2-overexpressing rice and wildtype plants. Our results revealed that the GS1;1-, GS1;2-overexpressing plants exhibited a poor plant growth phenotype and yield and decreased carbon/nitrogen ratio in the stem caused by the accumulation of nitrogen in the stem. In addition, the leaf SPAD value and photosynthetic parameters, soluble proteins and carbohydrates varied greatly in the GS1;1-, GS1;2-overexpressing plants. Furthermore, metabolite profile and gene expression analysis demonstrated significant changes in individual sugars, organic acids and free amino acids, and gene expression patterns in GS1;1-, GS1;2-overexpressing plants, which also indicated the distinct roles that these two GS1 genes played in rice nitrogen metabolism, particularly when sufficient nitrogen was applied in the environment. Thus, the unbalanced carbon-nitrogen metabolic status and poor ability of nitrogen transportation from stem to leaf in GS1;1-, GS1;2-overexpressing plants may explain the poor growth and yield. PMID:24743556

  5. Design, synthesis and biological evaluation of imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives as Mycobacterium tuberculosis pantothenate synthetase inhibitors.

    Science.gov (United States)

    Samala, Ganesh; Devi, Parthiban Brindha; Saxena, Shalini; Meda, Nikhila; Yogeeswari, Perumal; Sriram, Dharmarajan

    2016-03-15

    In the present study, we have designed imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives from earlier reported imidazo[1,2-a]pyridine based Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibitors. We synthesized thirty compounds and they were evaluated for MTB PS inhibition study, in vitro anti-TB activities against replicative and non-replicative MTB, in vivo activity using Mycobacterium marinum infected Zebra fish and cytotoxicity against RAW 264.7 cell line. Among them compound 2-methyl-N'-(4-phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bc) emerged as potent compound active against MTB PS with IC50 of 0.53±0.13 μM, MIC of 3.53 μM, 2.1 log reduction against nutrient starved MTB, with 33% cytotoxicity at 50 μM. It also showed 1.5 log reduction of M. marinum load in Zebra fish at 10mg/kg. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Mutations in LACS2, a Long-Chain Acyl-Coenzyme A Synthetase, Enhance Susceptibility to Avirulent Pseudomonas syringae But Confer Resistance to Botrytis cinerea in Arabidopsis1[OA

    Science.gov (United States)

    Tang, Dingzhong; Simonich, Michael T.; Innes, Roger W.

    2007-01-01

    We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea. PMID:17434992

  7. 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...

  8. Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

    Science.gov (United States)

    Campbell, S A; Alawa, J; Doro, B; Henriquez, F L; Roberts, C W; Nok, A; Alawa, C B I; Alsaadi, M; Mullen, A B; Carter, K C

    2012-02-08

    Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase.

    Science.gov (United States)

    Powers, S G; Meister, A

    1976-09-01

    The activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures. The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride. Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it. Such [14C]formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the [14C]formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme. When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of [14C]formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme. When reaction mixtures containing the enzyme, [gamma-32P]ATP, and [14C]bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate. Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate. Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.

  10. 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

  11. 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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

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

    Science.gov (United States)

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

    2004-03-26

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

  13. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans.

    Science.gov (United States)

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-03-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l(-1) when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24-30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  14. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans

    Science.gov (United States)

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-01-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24–30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis. PMID:24423427

  15. Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light

    International Nuclear Information System (INIS)

    Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

    1998-01-01

    The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition. (author)

  16. Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

    Science.gov (United States)

    Böttcher, Christine; Boss, Paul K.; Davies, Christopher

    2011-01-01

    Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes. PMID:21543520

  17. Regionally specific distribution of the binding of anti-glutamine synthetase and anti-S100 antibodies and of Datura stramonium lectin in glial domains of the optic lobe of the giant prawn.

    Science.gov (United States)

    Allodi, Silvana; Bressan, Cristine M; Carvalho, Sergio L; Cavalcante, Leny A

    2006-04-15

    We previously characterized some crustacean glial cells by markers such as 2',3'-cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein. Here we use antibodies against glutamine synthetase full-length molecule (anti-GS/FL), a GS C-terminal peptide (anti-GS/20aa-C), and brain S100 (anti-S100), as well as the binding of the insect glia and rat astrocytic marker Datura stramonium lectin (DSL), in the optic lobe of the prawn Macrobrachium rosenbergii. All markers label the lamina ganglionaris cartridge region (lighter: anti-GS/FL; heavier: DSL). In addition, anti-GS/FL labels superficial somata of external and internal medullas and internal chiasm cells. Both anti-GS/20aa-C and anti-S100 label heavily the glial sheaths of the lamina ganglionaris. In addition, anti-S100 binds to the perineurial glia of medullary parenchymal vessels. Western blot analyses show that both anti-GS/FL and anti-GS/20aa-C bind mostly to a band of 50-55 kDa, compatible with a long isoform of vertebrate GS, and accessorily to a possible dimer and, in the case of anti-GS/20aa-C, to an ill-defined band of intermediate mass. Binding of anti-S100 is selective for a single band of about 68 kDa but shows no protein in the weight range of the canonical S100 protein superfamily. DSL reveals two bands of about 75 and about 120 kDa, thus within the range of maximal recognition for rat astrocytes. Our results suggest that phenotype protein markers of the optic lobe glia share antigenic determinants with S100 and (a long form of) GS and that, similarly to vertebrate and insect glia, crustacean glia protein and N-glycan residue markers display regional heterogeneity.

  18. Association of anti-aminoacyl-transfer RNA synthetase antibody and anti-melanoma differentiation-associated gene 5 antibody with the therapeutic response of polymyositis/dermatomyositis-associated interstitial lung disease.

    Science.gov (United States)

    Yoshida, Naomi; Okamoto, Masaki; Kaieda, Shinjiro; Fujimoto, Kiminori; Ebata, Tomohiro; Tajiri, Morihiro; Nakamura, Masayuki; Tominaga, Masaki; Wakasugi, Daisuke; Kawayama, Tomotaka; Kuwana, Masataka; Mimori, Tsuneyo; Ida, Hiroaki; Hoshino, Tomoaki

    2017-01-01

    We attempted to clarify whether the presence of anti-aminoacyl-transfer RNA synthetase antibody (anti-ARS Ab) or anti-melanoma differentiation-associated gene 5 antibody (anti-MDA5 Ab) is associated with the therapeutic response of polymyositis/dermatomyositis-associated interstitial lung disease (PM/DM-ILD). We retrospectively investigated 22 patients with PM/DM-ILD (10 positive for anti-ARS Ab and nine positive for anti-MDA5 Ab) for whom antibody analysis of conserved serum was possible. We assessed mortality in the first three months as the therapeutic response in the acute phase and compared changes in clinical data for up to one year considered as the chronic phase. We classified the clinical changes over the year into three groups: Improvement (increased % vital capacity [%VC] or diffusing capacity of the lung for carbon monoxide [%D LCO ]≥10 or 15%), deterioration (decreased %VC or %D LCO ≥10 or 15%), and no change (remainder of the changes). The extent of abnormality demonstrated by high-resolution computed tomography (HRCT) was scored. Positivity for anti-MDA5 Ab, but not for anti-ARS Ab, was associated with mortality in the first 3 months. Evaluation of the therapeutic response in the first year showed that positivity for the anti-ARS Ab, but not for the anti-MDA5 Ab, was associated with an improvement in %D LCO and a decline in the serum KL-6 levels. Positivity for the anti-ARS Ab or negativity for anti-MDA5 Ab was associated with a greater decrease in bronchial dilatation as seen by HRCT. Anti-ARS and anti-MDA5 Abs are associated with the therapeutic response of PM/DM-ILD. Copyright © 2016. Published by Elsevier B.V.

  19. Combined use of heat-shock protein 70 and glutamine synthetase is useful in the distinction of typical hepatocellular adenoma from atypical hepatocellular neoplasms and well-differentiated hepatocellular carcinoma

    Science.gov (United States)

    Nguyen, Thuy B; Roncalli, Massimo; Tommaso, Luca Di; Kakar, Sanjay

    2017-01-01

    Well-differentiated hepatocellular carcinoma can mimic high-grade dysplastic nodule in cirrhotic liver and hepatocellular adenoma in non-cirrhotic liver. This study evaluates the efficacy of combined use of heat-shock protein 70 (HSP70), glutamine synthetase (GS) and glypican-3 in this setting. Immunohistochemistry for these three markers was done in 17 typical hepatocellular adenoma, 15 high-grade dysplastic nodules, 20 atypical hepatocellular neoplasms (14 clinically atypical and 6 pathologically atypical), 14 very well-differentiated hepatocellular carcinoma, and 43 well-differentiated hepatocellular carcinoma. All three markers were negative in typical adenomas. HSP70 was positive in 10, 71, and 67% of atypical neoplasms, very well-differentiated and well-differentiated HCC, respectively, while GS was positive in 60, 50, and 60% of atypical neoplasms, very well-differentiated and well-differentiated hepatocellular carcinoma, respectively. Glypican-3 was negative in all atypical neoplasms and very well-differentiated hepatocellular carcinoma, and was positive in 27% of well-differentiated hepatocellular carcinoma. Positive staining with at least one marker (HSP70 and/or GS) was seen in 85% of very well-differentiated hepatocellular carcinoma, which was similar to well-differentiated hepatocellular carcinoma (78%, P = 0.4), and pathologically atypical cases (100%, P = 0.5), but significantly higher compared with clinically atypical cases (43%. P = 0.03) and none of typical adenomas (P hepatocellular carcinoma compared with atypical neoplasms (45 vs 10%, P = 0.004). Both these markers were also more often expressed in very well-differentiated hepatocellular carcinoma compared with atypical cases (38 vs 10%, P = 0.06). In conclusion, the combined use of GS and HSP70 can be useful in the diagnosis of very well-differentiated hepatocellular carcinoma. These stains can also help in the distinction of typical adenoma from atypical hepatocellular neoplasms. Glypican-3

  20. Combined use of heat-shock protein 70 and glutamine synthetase is useful in the distinction of typical hepatocellular adenoma from atypical hepatocellular neoplasms and well-differentiated hepatocellular carcinoma.

    Science.gov (United States)

    Nguyen, Thuy B; Roncalli, Massimo; Di Tommaso, Luca; Kakar, Sanjay

    2016-03-01

    Well-differentiated hepatocellular carcinoma can mimic high-grade dysplastic nodule in cirrhotic liver and hepatocellular adenoma in non-cirrhotic liver. This study evaluates the efficacy of combined use of heat-shock protein 70 (HSP70), glutamine synthetase (GS) and glypican-3 in this setting. Immunohistochemistry for these three markers was done in 17 typical hepatocellular adenoma, 15 high-grade dysplastic nodules, 20 atypical hepatocellular neoplasms (14 clinically atypical and 6 pathologically atypical), 14 very well-differentiated hepatocellular carcinoma, and 43 well-differentiated hepatocellular carcinoma. All three markers were negative in typical adenomas. HSP70 was positive in 10, 71, and 67% of atypical neoplasms, very well-differentiated and well-differentiated HCC, respectively, while GS was positive in 60, 50, and 60% of atypical neoplasms, very well-differentiated and well-differentiated hepatocellular carcinoma, respectively. Glypican-3 was negative in all atypical neoplasms and very well-differentiated hepatocellular carcinoma, and was positive in 27% of well-differentiated hepatocellular carcinoma. Positive staining with at least one marker (HSP70 and/or GS) was seen in 85% of very well-differentiated hepatocellular carcinoma, which was similar to well-differentiated hepatocellular carcinoma (78%, P=0.4), and pathologically atypical cases (100%, P=0.5), but significantly higher compared with clinically atypical cases (43%. P=0.03) and none of typical adenomas (Phepatocellular carcinoma compared with atypical neoplasms (45 vs 10%, P=0.004). Both these markers were also more often expressed in very well-differentiated hepatocellular carcinoma compared with atypical cases (38 vs 10%, P=0.06). In conclusion, the combined use of GS and HSP70 can be useful in the diagnosis of very well-differentiated hepatocellular carcinoma. These stains can also help in the distinction of typical adenoma from atypical hepatocellular neoplasms. Glypican-3 has low

  1. Acyl-CoA synthetase short-chain family member 2 (ACSS2) is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells.

    Science.gov (United States)

    Xu, Huifen; Luo, Jun; Ma, Gongzhen; Zhang, Xueying; Yao, Dawei; Li, Ming; Loor, Juan J

    2018-02-01

    Sterol regulatory element binding protein 1 (SREBP-1) is well-known as the master regulator of lipogenesis in rodents. Acyl-CoA synthetase short-chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl-CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl-CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non-lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p < 0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p < 0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP-1. Furthermore, SREBP-1 interacted with an SRE (SREBP response element) spanning at -475 to -483 bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP-1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2. © 2017 Wiley Periodicals, Inc.

  2. Nitrate reductase and glutamine synthetase activity in coffee leaves during fruit development Atividade da redutase do nitrato e glutamina sintetase em folhas de cafeeiro durante o desenvolvimento dos frutos

    Directory of Open Access Journals (Sweden)

    Andre Rodrigues Reis

    2009-04-01

    Full Text Available Nitrate reductase is the first enzyme in the pathway of nitrate reduction by plants, followed by glutamine synthetase, which incorporates ammonia to glutamine. The purpose of this study was to evaluate the nitrate reductase and glutamine synthetase activity, total soluble protein content, N and Ni content in coffee leaves during fruit development under field conditions to establish new informations to help assess the N nutritional status and fertilizer management. The experimental design was in randomized complete blocks, arranged in a 3 x 6 factorial design, with five replications. The treatments consisted of 3 N rates (0 - control, 150 and 300 kg ha-1 and six evaluation periods (January, February, March, April, May, and June in six-year-old coffee (Coffea arabica L. plants of Catuaí Vermelho IAC 44 cv. The nitrate reductase and glutamine synthetase activities, leaf soluble protein, and N concentrations increased linearly with the N rates. During fruit development, the enzyme activity, leaf soluble protein and N content decreased, due to the leaf senescence process caused by nutrient mobilization to other organs, e.g, to the berries. Leaf Ni increased during fruit development. Beans and raisin-fruits of plants well-supplied with N had higher Ni contents. Enzyme activities, total leaf N and leaf soluble protein, evaluated during the green fruit stage in March, were significantly correlated with coffee yield. These variables can therefore be useful for an early assessment of the coffee N nutritional status as well as coffee yield and N fertilization management.A redutase do nitrato (RN é a primeira enzima na via de redução de nitrato pelas plantas, seguida da glutamina sintetase (GS, a qual incorpora amônia à glutamina. O objetivo deste trabalho foi avaliar a atividade dessas enzimas, o teor de proteína solúvel total e a concentração de N e de Ni em folhas e grãos de cafeeiro durante o desenvolvimento dos frutos, em condições de

  3. rs657075 (CSF2) Is Associated with the Disease Phenotype (BAS-G) of Ankylosing Spondylitis

    Science.gov (United States)

    Chen, Wei-Chiao; Wei, James Cheng-Chung; Lu, Hsing-Fang; Wong, Henry Sung-Ching; Woon, Peng Yeong; Hsu, Yu-Wen; Huang, Jin-Ding; Chang, Wei-Chiao

    2017-01-01

    Ankylosing spondylitis (AS) is a systemic autoimmune disease mainly affecting the lumbar spine and sacroiliac joints, and exhibits peripheral inflammatory arthropathy. More than 25 loci have been identified as associated with AS. Because both AS and rheumatoid arthritis (RA) are autoimmune diseases that may share some common genetic factors, we therefore examined if the newly identified RA genetic polymorphisms were associated with AS in a Taiwanese population. In this study, we enrolled 475 AS patients and 11,301 healthy subjects from a Taiwanese biobank as controls. Although none of single-nucleotide polymorphisms (SNPs) were associated with the susceptibility to AS, the AS disease index Bath AS Global (BAS-G) clinical phenotype was observed as significantly correlated to the AA genotype of rs657075 (CSF2). The significance remains after gender/age/disease duration adjustment and after group categorization by human leukocyte antigen-B 27 (HLA-B27) genotype. We further investigated the possible functions of rs657075 through bioinformatics approaches. Results revealed that polymorphism of rs657075 is able to influence the expression of acyl-CoA synthetase long-chain family member 6 (ACSL6). In conclusion, our study indicated that rs657075 (CSF2) is strongly associated with the AS disease index Bath AS Global (BAS-G) clinical phenotype. PMID:28054948

  4. The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Jin, Xiaofen; Peng, Yanyan; Wang, Meng; Liu, Hao; Liu, Xiaoling; Zhang, Zengjun; Ji, Yanchun; Zhang, Juanjuan; Liang, Min; Zhao, Fuxin; Sun, Yan-Hong; Zhang, Minglian; Zhou, Xiangtian; Chen, Ye; Mo, Jun Qin; Huang, Taosheng; Qu, Jia; Guan, Min-Xin

    2016-02-01

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2. © The Author 2015. Published by Oxford University Press

  5. Genetics Home Reference: glutathione synthetase deficiency

    Science.gov (United States)

    ... slowing down of physical reactions, movements, and speech (psychomotor retardation); intellectual disability; and a loss of coordination ( ... occur? How can gene mutations affect health and development? More about Mutations and Health Inheritance Pattern This ...

  6. Evolutionary relationships of the carbamoylphosphate synthetase genes

    NARCIS (Netherlands)

    van den Hoff, M. J.; Jonker, A.; Beintema, J. J.; Lamers, W. H.

    1995-01-01

    Carbamoylphosphate is a common intermediate in the metabolic pathways leading to the biosynthesis of arginine and pyrimidines. The amino acid sequences of all available proteins that catalyze the formation of carbamoylphosphate were retrieved from Genbank and aligned to estimate their mutual

  7. Evolutionary relationships of the carbamoylphosphate synthetase genes

    NARCIS (Netherlands)

    vandenHoff, MJB; Jonker, A; Beintema, JJ; Lamers, WH

    1995-01-01

    Carbamoylphosphate is a common intermediate in the metabolic pathways leading to the biosynthesis of arginine and pyrimidines, The amino acid sequences of all available proteins that catalyze the formation of carbamoylphosphate were retrieved from Genbank and aligned to estimate their mutual

  8. Engineering polyketide synthases and nonribosomal peptide synthetases.

    Science.gov (United States)

    Williams, Gavin J

    2013-08-01

    Naturally occurring polyketides and nonribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and nonribosomal peptide biosynthetic machinery. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Enhancement of glutamine synthetase activity in Paenibacillus ...

    African Journals Online (AJOL)

    Accession No AB727983). High GS activity was recorded in the two strains, in presence of the divalent cations Mg+2 and Mn+2. Western blot analysis confirmed the presence of the GS at approximately ~60 kDa. GS activity was found to be affected by ...

  10. Lincosamide Synthetase-A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

    Czech Academy of Sciences Publication Activity Database

    Janata, Jiří; Kadlčík, Stanislav; Koběrská, Markéta; Ulanová, Dana; Kameník, Zdeněk; Novák, Petr; Kopecký, Jan; Novotná, Jitka; Radojevič, Bojana; Plháčková, Kamila; Gažák, Radek; Najmanová, Lucie

    2015-01-01

    Roč. 10, č. 3 (2015) E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : BIOSYNTHETIC GENE-CLUSTER * MOLECULAR-WEIGHT THIOL * PHOSPHOPANTETHEINYL TRANSFERASE Subject RIV: EE - Microbiology, Virology Impact factor: 3.057, year: 2015

  11. Acyl-CoA synthetase long-chain 5 genotype is associated with body composition changes in response to lifestyle interventions in postmenopausal women with overweight and obesity: a genetic association study on cohorts Montréal-Ottawa New Emerging Team, and Complications Associated with Obesity.

    Science.gov (United States)

    Rajkumar, Abishankari; Lamothe, Gilles; Bolongo, Pierrette; Harper, Mary-Ellen; Adamo, Kristi; Doucet, Éric; Rabasa-Lhoret, Remi; Prud'homme, Denis; Tesson, Frédérique

    2016-08-11

    Genetic studies on Acyl-CoA Synthetase Long-Chain 5 (ACSL5) demonstrate an association between rs2419621 genotype and rate of weight loss in women with obesity in response to caloric restriction. Our objectives were to (1) confirm results in two different populations of women with overweight and obesity (2) study rs2419621's influence on body composition parameters of women with overweight and obesity following lifestyle interventions. rs2419621 genotype was determined in women with overweight and obesity who participated in the Montréal-Ottawa New Emerging Team (MONET n = 137) and Complications Associated with Obesity (CAO n = 37) studies. Genotyping was done using TaqMan MGB probe-based assay. Multiple linear regression analyses were used to test for associations. When studying women with overweight and obesity, rs2419621 [T] allele carriers had a significantly greater decrease in visceral fat, absolute and percent fat mass and a greater increase in percent lean mass in response to lifestyle intervention in comparison to non-carriers. Studying only individuals with obesity showed similar results with rs2419621 [T] allele carriers also displaying a significantly greater decrease in body mass index following the lifestyle intervention in comparison to non-carriers. Women with overweight and obesity carrying the ACSL5 rs2419621 [T] allele are more responsive to lifestyle interventions in comparison to non-carriers. Conducting such genetic association studies can aid in individualized treatments/interventions catered towards an individual's genotype.

  12. The murine double-stranded RNA-dependent protein kinase PKR and the murine 2',5'-oligoadenylate synthetase-dependent RNase L are required for IFN-β-mediated resistance against herpes simplex virus type 1 in primary trigeminal ganglion culture

    International Nuclear Information System (INIS)

    Al-khatib, Khaldun; Williams, Bryan R.G.; Silverman, Robert H.; Halford, William; Carr, Daniel J.J.

    2003-01-01

    A study was undertaken to evaluate the efficacy of an adenoviral construct expressing the murine interferon-β (IFN-β) transgene (Ad:IFN-β) against herpes simplex virus type 1 (HSV-1) infection in a primary trigeminal ganglion (TG) cell culture. The transduction efficiency ranged from 0.2 to 11.0% depending on the multiplicity of infection (m.o.i.) of the adenoviral vector (0.5-50.0). Moreover, neurons were the main target of the adenoviral transduction. TG cultures transduced with Ad:IFN-β displayed up to a 19-fold reduction in viral titers compared with cells transduced with an Ad:Null or nontransduced TG culture controls. Transduction with Ad:IFN-β up-regulated two critical antiviral genes, double-stranded RNA-dependent protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS). The absence of PKR or RNase L (downstream effector molecule of OAS) attenuated Ad:IFN-β efficacy against HSV-1 replication, implicating a critical role for PKR and OAS/RNase systems in the establishment of IFN-induced resistance against HSV-1 in TG cells

  13. ß-Lysine discrimination by lysyl-tRNA synthetase

    DEFF Research Database (Denmark)

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

    2011-01-01

    guided by the PoxA structure. A233S LysRS behaved as wild type with a-lysine, while the G469A and A233S/G469A variants decreased stable a-lysyl-adenylate formation. A233S LysRS recognized ß-lysine better than wildtype, suggesting a role for this residue in discriminating a- and ß-amino acids. Both...

  14. Glutamine synthetase expression in perinatal spiny mouse liver

    NARCIS (Netherlands)

    Lamers, W. H.; Boon, L.; van Hemert, F. J.; Labruyère, W. T.; de Jong, P.; Ruijter, J. M.; Moorman, A. F.

    1999-01-01

    The pronounced increase in the protein/mRNA ratio of ammonia-metabolising enzymes in rat liver in the last prenatal week represents a clear example of a post-transcriptional level of control of gene expression. Both the underlying mechanism, namely an increase in translational efficiency of the mRNA

  15. Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

    NARCIS (Netherlands)

    Bosveld, Floris; Rana, Anil; Lemstra - Wierenga, Willemina; Kampinga, Harm; Sibon, Ody

    2008-01-01

    Background: Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in

  16. Prostaglandin synthetase activity of goat vesicular microsomes: Co ...

    Indian Academy of Sciences (India)

    tribpo

    The homogenate was centrifuged for 10 min at 12,000 g in a Sorvall Model. RC 5B centrifuge. The supernatant fraction was passed through a double layer of cheese cloth and centrifuged again at 100,000 g for 1 h in a Beckman Ultracentrifuge. Model L5-50. The resulting pellet was suspended in 2 volumes of 0.1 Μ tris-HCl.

  17. 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...

  18. An oxalyl-CoA synthetase is important for oxalate metabolism in Saccharomyces cerevisiae

    Science.gov (United States)

    Although oxalic acid is common in nature, our understanding of the mechanism(s) regulating its turnover remains incomplete. In this study we identify Saccharomyces cerevisiae acyl-activating enzyme 3 (ScAAE3) as an enzyme capable of catalyzing the conversion of oxalate to oxalyl-CoA. Based on our fi...

  19. 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.

  20. Hexose phosphate synthetase from Methylococcus capsulatus makes d-arabino-3-hexulose phosphate

    Science.gov (United States)

    Kemp, M. B.

    1974-01-01

    The product of the reaction catalysed by hexose phosphate synthase prepared from Methylococcus capsulatus was dephosphorylated and the sugar moiety purified. The sugar and derivatives were compared by various chromatographic and other methods with authentic samples of allulose (psicose), d-erythro-l-glycero-3-hexulose and d-erythro-d-glycero-3-hexulose. The sugar is not allulose, as was previously thought on the basis of less extensive evidence (Kemp & Quayle, 1966), but is in fact d-erythro-l-glycero-3-hexulose (d-arabino-3-hexulose). This identification is consistent with recent studies which have shown that hexose phosphate synthase catalyses the condensation of formaldehyde with d-ribulose 5-phosphate rather than with d-ribose 5-phosphate (Kemp, 1972). PMID:4463938

  1. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... of molecular biology were thinking on gene expression and genetic code. In a famous letter send in 1955 to the “RNA Tie. Club” Francis Crick predicted the existence of small adaptor. RNA molecules that would carry their own amino acids and. The early history of tRNA recognition by aminoacyl-tRNA.

  2. 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 Access to a series of truncated ATP analogs, as potential anti-tuberculosis agents, has been explored via alkylation and acylation of 3-aminophenol, whereas chloroacetylation, using chloroacetyl chloride, and subsequent Arbuzov phosphonation of a...

  3. Gene Regulation by Metabolic Enzyme GMP Synthetase and Chromatin Remodeler NuRD

    NARCIS (Netherlands)

    B.A. Reddy (Ashok)

    2012-01-01

    textabstractSpatial and temporal control of the gene expression is crucial for normal growth and development of an organism. Environmental stress factors pose a constant threat to normal development of an organism by causing altered gene expression. Cells have evolved counteractive mechanisms to

  4. 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

    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...

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

    Czech Academy of Sciences Publication Activity Database

    Juguet, M.; Lautru, S.; Francou, F.-X.; Nezbedová, Šárka; Leblond, P.; Gondry, M.; Pernodet, J. L.

    2009-01-01

    Roč. 16, č. 4 (2009), s. 421-431 ISSN 1074-5521 Institutional research plan: CEZ:AV0Z50200510 Keywords : ADENYLATION DOMAINS * ESCHERICHIA-COLI * FUNCTIONAL-ANALYSIS Subject RIV: CE - Biochemistry Impact factor: 6.523, year: 2009

  6. Nuclear actin aggregation is a hallmark of anti-synthetase syndrome-induced dysimmune myopathy

    NARCIS (Netherlands)

    Stenzel, Werner; Preuße, Corinna; Allenbach, Yves; Pehl, Debora; Junckerstorff, Reimar; Heppner, Frank L.; Nolte, Kay; Aronica, Eleonora; Kana, Veronika; Rushing, Elisabeth; Schneider, Udo; Claeys, Kristl G.; Benveniste, Olivier; Weis, Joachim; Goebel, Hans H.

    2015-01-01

    To analyze antisynthetase syndrome-associated myositis by modern myopathologic methods and to define its place in the spectrum of idiopathic inflammatory myopathies (IIMs). Skeletal muscle biopsies from antisynthetase syndrome-associated myositis and other IIMs from different institutions worldwide

  7. Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Cairns, Timothy; Stack, Deirdre

    2011-01-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricro...

  8. Gene polymorphism of aldosterone synthetase (CYP11B2 variants and main cardiovascular risk factors

    Directory of Open Access Journals (Sweden)

    L. Ye. Lobach

    2016-12-01

    Full Text Available Background. Purpose of the work – to investigate the possible relationship of the cardiovascular risk main factors with certain polymorphism of aldosterone synthase gene (CYP11B2. Materials and methods. Аt the Cardiology Department of PL Shupyk NMAPE general clinical examination of 378 patients was held. Patients were divided into four groups: 100 patients with postinfarction cardiosclerosis, 78 patients with CAD without myocardial infarction in history, 100 high cardiovascular risk patients (with diabetes, hypertension or dyslipidemia and 100 healthy patients (absence of cardiovascular disease was confirmed by medical history, ECG, blood pressure measurement and stress-ECG. Genetic testing was performed by polymerase chain reaction in real time at the Institute of Physiology named after O. O. Bogomolets. Exclusion criteria were hemodynamically significant valvular heart disease, chronic obstructive pulmonary disease, permanent or temporary heart pacing, acute heart failure and implanted cardioverter-defibrillator, permanent form of atrial fibrillation. Statistical analysis of the results was performed using Microsoft Excel, the statistical program SPSS (version 13US. Results. When analyzing the average levels of low density lipoprotein (LDL cholesterol statistically significant difference between the group of patients with postinfarction cardiosclerosis and the group of high-risk patients (2.93±1.2 mmol/L vs 3.4±1.2 mmol/L, p=0.0075 was demonstrated, indicating a better cholesterol control in the group of patients with postinfarction cardiosclerosis, despite the fact that the average cholesterol level did not reach the target. The highest average levels of triglycerides (TG were observed in patients with postinfarction cardiosclerosis – 1.56±0.725 mmol/L, intermediate – in patients with stable coronary artery disease – 1.39±0.795 mmol/L, and the lowest – in high cardiovascular risk patients – 1.04±0.565 mmol/L, with significant differences between all groups. The level of total cholesterol in patients with postinfarction cardiosclerosis was significantly higher in the subgroup of patients with homozygous recessive variant CC (5.8±1.08 mmol/L, compared with heterozygotes TC (4.87±1.3 mmol/L, p=0.024 and had no statistical significance when compared with the dominant TT homozygotes (5.06±1.45 mmol. Higher levels of total cholesterol (5.76±1.5 mmol/L in TT homozygotes compared with TC (4.92±1.27 mmol/L, p=0.027 and CC (4.74±1.23 mmol/L, p=0.022 were found. In the group of patients with postinfarction cardiosclerosis, LDL cholesterol in the subgroup homozygous recessive variant CC was higher (3.43±0.87 mmol/L (not significant compared with a TT variant (3.02±1.3 mmol/L and compared with heterozygotes TC (2.78±1.2 mmol/L, p=0.08. The level of TG in patients with stable coronary heart disease was the lowest in dominant homozygotes TT (1.13±0.56 mmol/L compared with CT heterozygotes (1.54±0.97 mmol/L, p=0.08 and CC homozygotes (1.36±0.58 mmol/L, p=0.2. Conclusions. 1. Group of high cardiovascular risk patients requires special attention in the prevention of cardiovascular diseases, because this group showed the worst control of cardiovascular risk factors (level of total cholesterol, LDL cholesterol, glucose, SBP in the absence of appropriate treatment. 2. Ingroup of patients with stable coronary artery disease and postinfarction cardiosclerosis the link CC variant gene polymorphism of aldosterone synthase CYP11B2-344C/T with higher levels of total cholesterol, LDL cholesterol was established, which increases the cardio-vascular risk in this group. 3. CC polymorphism of aldosterone synthase gene CYP11B2-344C/T was associated with higher levels of SBP in patients with stable coronary artery disease and postinfarction cardiosclerosis, which increases the cardiovascular risk such as the development of hypertension.

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

    DEFF Research Database (Denmark)

    Guan, Miao

    Nitrogen is one of the major plant nutrients limiting crop production worldwide. In many parts of the world the availability of N fertilizers is limited, whereas in other parts of the world too much N fertilizer is applied, leading to serious negative environmental consequences. The use of N...... but primary root development during seed germination was reduced in the presence of external N. Gln1;2 promoter-GFP construct showed that Gln1;2 expression was localized to the vascular cells of roots, petals, and stamens. A novel compensatory interaction between Gln1;1 and Gln1;2 was shown since shoot Gln1...

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

    NARCIS (Netherlands)

    Meussen, B.J.; Weusthuis, R.A.; Sanders, J.P.M.; Graaff, de L.H.

    2012-01-01

    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,

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

    DEFF Research Database (Denmark)

    Guan, Miao

    fertilizers accordingly needs to be optimized in order to make agriculture more sustainable. One pathway to achieve such optimization is to improve plant N use efficiency (NUE) by developing new crop genotypes with improved yield per unit of N fertilizer applied. For this purpose, more and better knowledge......Nitrogen is one of the major plant nutrients limiting crop production worldwide. In many parts of the world the availability of N fertilizers is limited, whereas in other parts of the world too much N fertilizer is applied, leading to serious negative environmental consequences. The use of N...... about bottlenecks in plant N assimilation is needed. Based on a reverse genetics strategy embracing characterization of knockout mutants in the model plant species Arabidopsis, the results obtained in this PhD study have provided new information about the specific roles of two genes Gln1;1 and Gln1...

  12. Emerging Signaling Pathway in Arcuate Feeding-Related Neurons: Role of the Acbd7

    Directory of Open Access Journals (Sweden)

    Damien Lanfray

    2017-06-01

    Full Text Available The understanding of the mechanisms whereby energy balance is regulated is essential to the unraveling of the pathophysiology of obesity. In the last three decades, focus was put on the metabolic role played by the hypothalamic neurons expressing proopiomelanocortin (POMC and cocaine and amphetamine regulated transcript (CART and the neurons co-localizing agouti-related peptide (AgRP, neuropeptide Y (NPY, and gamma-aminobutyric acid (GABA. These neurons are part of the leptin-melanocortin pathway, whose role is key in energy balance regulation. More recently, the metabolic involvement of further hypothalamic uncharacterized neuron populations has been suggested. In this review, we discuss the potential homeostatic implication of hypothalamic GABAergic neurons that produce Acyl-Coa-binding domain containing protein 7 (ACBD7, precursor of the nonadecaneuropeptide (NDN, which has recently been characterized as a potent anorexigenic neuropeptide capable of relaying the leptin anorectic/thermogenic effect via the melanocortin system.

  13. 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

    , contributed significantly to the clearance of parasites with multiple dhfr/dhps mutations. However, these mutations have a survival advantage as they were associated with increased gametocytogenesis. The above implications of dhfr/dhps mutations were associated with MOM 2 to 5, regardless of the gene...

  14. Inositol-1-phosphate synthetase mRNA as a new target for antisense inhibition of Mycobacterium tuberculosis.

    Science.gov (United States)

    Li, Yuanyuan; Chen, Zhifei; Li, Xiaobo; Zhang, Hongling; Huang, Qiang; Zhang, Ying; Xu, Shunqing

    2007-03-10

    The need for novel antimicrobial agents to combat the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis is a worldwide urgency. This study has investigated the effects on phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of inositol-1-phosphate synthase, the key enzyme in the first step in inositol synthesis. Inositol is utilized by M. tuberculosis in the production of its major thiol, which is an antioxidant that helps M. tuberculosis to get rid of reactive oxygen species and electrophilic toxins. Real-time RT-PCR analysis revealed that mRNA expression of inositol-1-phosphate (I-1-P) synthase was significantly reduced upon addition of 20 microM PS-ODNs. Treatment with antisense PS-ODNs also reduced the level of mycothiol and the proliferation of M. tuberculosis and enhanced susceptibility to antibiotics. The experiments indicated that the antisense PS-ODNs could enter the cytoplasm of M. tuberculosis and inhibit the expression of I-1-P synthase. This study demonstrates that the M. tuberculosis I-1-P synthase is a target for the development of novel antibiotics and PS-ODN to I-1-P synthase is a promising antimycobaterial candidate.

  15. Prevention of Endotoxin-Induced Pulmonary Hypertension in Primates by the Use of a Selective Thromboxane Synthetase Inhibitor, OXY 1581

    Science.gov (United States)

    1982-09-01

    ZBI). Arterial and mixed venous blood al., 1981; Schiantarelli et al., 1981; Altura and Chand, 1981). gases were measured on a blood gas analyzer...HARRIS, R., ZMUDKA, M., MADDOX. Y., RAMWELL, P. AND FLETCHER, J. R.: S Relationship of TxB2 and 6-keto-PGFIa to the hemodynamic changes during ALTURA , B

  16. 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 requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame......, showing a high degree of similarity to glutathione peroxidases from other organisms was identified....

  17. Phosphoribosylpyrophosphate synthetase superactivity and recurrent infections is caused by a p.Val142Leu mutation in PRS-I

    NARCIS (Netherlands)

    Moran, Rocio; van Kuilenburg, André B. P.; Duley, John; Nabuurs, Sander B.; Retno-Fitri, Aditia; Christodoulou, John; Roelofsen, Jeroen; Yntema, Helger G.; Friedman, Neil R.; van Bokhoven, Hans; de Brouwer, Arjan P. M.

    2012-01-01

    We identified a novel missense mutation, c.424G>C (p.Val142Leu) in PRPS1 in a patient with uric acid overproduction without gout but with developmental delay, hypotonia, hearing loss, and recurrent respiratory infections. The uric acid overproduction accompanying this combination of symptoms

  18. 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

    The diploid Lotus japonicus was previously suggested as a model for the legume plant family. We present here the nucleotide sequence and the derived gene organization of a small part of the genome in this model plant. Two functional genes with the same transcriptional orientation were identified...

  19. 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

    and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression...... 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...... and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1(H-/-) hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1(H...

  20. Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1996-01-01

    reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth...