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Sample records for acyl-coa synthetase fadd13

  1. Dissecting the role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis.

    Khare, Garima; Gupta, Vibha; Gupta, Rakesh K; Gupta, Radhika; Bhat, Rajiv; Tyagi, Anil K

    2009-01-01

    Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the

  2. Dissecting the role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13 of Mycobacterium tuberculosis.

    Garima Khare

    Full Text Available Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb severely limit the treatment options for tuberculosis (TB; hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089 belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487 resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172 and Ala(302 to be involved in the binding of ATP and Ser(404 in the binding of CoenzymeA. The influence of mutations of the residues Val(209 and Trp(377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26/C(24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172, Ala(302 and Lys(487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that

  3. DISTINCT TRANSCRIPTIONAL REGULATION OF LONG-CHAIN ACYL-COA SYNTHETASE ISOFORMS AND CYTOSOLIC THIOESTERASE 1 IN THE RODENT HEART BY FATTY ACIDS AND INSULIN

    The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs)...

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

    Jules A Nchoutmboube

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

  5. Cytosolic glutamine synthetase

    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...

  6. Liver fatty acid binding protein (LFABP) transfers fatty acids and fatty acyl coas to membranes

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C.; Córsico, Betina

    2010-01-01

    The objective of this work was to analyze LFABP´s capacity to transfer acyl CoAs to artificial membranes and compare it to LCFA transfer employing natural ligands, in order to better understand the specific physiological role of LFABP in the cell.

  7. Cytosolic glutamine synthetase in barley

    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...

  8. Substrate specificity of formylglycinamidine synthetase

    Schendel, F.J.; Stubbe, J.

    1986-04-22

    Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. (Buchanan, J. M., Ohnoki, S., and Hong, B. S. (1978) Methods Enzymol. 51, 193-201) (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH/sub 2/NHCHO) has been replaced by R = CH/sub 3/, CH/sub 2/OH, CH/sub 2/Cl, CH/sub 2/NH/sub 3/, CH/sub 2/NHCOCH/sub 3/, CH/sub 2/NHCOCH/sub 2/Cl, CH/sub 2/NHCO/sub 2/CH/sub 2/Ph, and L-CHC-H/sub 3/NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH/sub 3/, CH/sub 2/OH, and CH/sub 2/NHCOCH/sub 3/ and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH/sub 2/NHCOCH/sub 3/ causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH/sub 2/NHCHO) and the FGAR analogue (R = CH/sub 2/NHCHOCH/sub 3/) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. (18O)-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.

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

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek; Marcher, Ann-Britt; Elle, Ida C; Helledie, Torben; Due, Marianne; Pagmantidis, Vasileios; Finsen, Bente; Wilbertz, Johannes; Kruhoeffer, Mogens; Faergeman, Nils; Mandrup, Susanne

    2011-01-01

    , little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice go through a crisis with overall weakness, and a slightly decreased growth...... rate. Using microarray analysis we show that the liver of ACBP-/- mice display a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The...... delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to...

  10. Radioimmune assay of human platelet prostaglandin synthetase

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH2 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 [125I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [125I]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 109 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

  11. Genetics Home Reference: glutathione synthetase deficiency

    ... elevated acidity in the blood and tissues (metabolic acidosis). In addition to the features present in moderate ... Kaabachi N, Hachicha M. Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency. Fetal Pediatr Pathol. ...

  12. Genetics Home Reference: holocarboxylase synthetase deficiency

    ... the body is unable to use the vitamin biotin effectively. This disorder is classified as a multiple ... impaired activity of certain enzymes that depend on biotin. The signs and symptoms of holocarboxylase synthetase deficiency ...

  13. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with (32P)orthophosphate

  14. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    Pendergast, A.M.

    1986-01-01

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with (/sup 32/P)orthophosphate.

  15. Neural control of glutamine synthetase activity in rat skeletal muscles.

    Feng, B; Konagaya, M; Konagaya, Y; Thomas, J W; Banner, C; Mill, J; Max, S R

    1990-05-01

    The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighted, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased approximately twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoid-mediated glutamine synthetase induction previously described by us. PMID:1970709

  16. Aromatase inhibitors and anti-synthetase syndrome.

    Mascella, Fabio; Gianni, Lorenzo; Affatato, Alessandra; Fantini, Manuela

    2016-09-01

    Adjuvant therapy in postmenopausal women with endocrine-responsive breast cancer (BC) is actually centered on the use of anti-aromatase inhibitors (AI). Several reports, however, are emerging in literature associating the use of this drugs to rheumatic disorders. This case report describes the first case of anti-synthetase syndrome diagnosis after treatment with anti-estrogen agents in a patient with pre-existing rheumatoid arthritis. PMID:27225465

  17. Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

    Zhao, Ming-Wei; Zhu, Bin; Hao, Rui; Xu, Min-Gang; Eriani, Gilbert; Wang, En-Duo

    2005-01-01

    The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (αβ-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNALeu and minihelixLeu. Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplan...

  18. Biosynthetic engineering of nonribosomal peptide synthetases.

    Kries, Hajo

    2016-09-01

    From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27465074

  19. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions carbamoyl phosphate synthetase I deficiency ...

  20. Continuous spectrophotometric assay for aminoacyl-tRNA synthetases.

    Chang, G G; Pan, F; Lin, Y H; Wang, H Y

    1984-11-01

    A simple, continuous assay for aminoacyl-tRNA synthetases utilizing a commercially available pyrophosphate assay reagent kit was demonstrated. The method coupled aminoacyl-tRNA synthetase activity with pyrophosphate-dependent fructose-6-phosphate kinase, aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase. PPi formation was correlated with the oxidation of NADH, and was monitored continuously by the decrease of absorbance at 340 nm. PMID:6099060

  1. Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii.

    Fox, Barbara A; Bzik, David J

    2003-01-01

    Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase. PMID:12547350

  2. Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases.

    Sun, Xun; Li, Hao; Alfermann, Jonas; Mootz, Henning D; Yang, Haw

    2014-12-23

    Nonribosomal peptide synthetases (NRPS) incorporate assorted amino acid substrates into complex natural products. The substrate is activated via the formation of a reactive aminoacyl adenylate and is subsequently attached to the protein template via a thioester bond. The reactive nature of such intermediates, however, leads to side reactions that also break down the high-energy anhydride bond. The off-pathway kinetics or their relative weights compared to that of the on-pathway counterpart remains generally elusive. Here, we introduce multiplatform kinetics profiling to quantify the relative weights of on- and off-pathway reactions. Using the well-defined stoichiometry of thioester formation, we integrate a mass spectrometry (MS) kinetics assay, a high-performance liquid chromatography (HPLC) assay, and an ATP-pyrophosphate (PPi) exchange assay to map out a highly efficient on-pathway kinetics profile of the substrate activation and intermediate uploading (>98% relative weight) for wide-type gramicidin S synthetase A (GrsA) and a 87% rate profile for a cysteine-free GrsA mutant. Our kinetics profiling approach complements the existing enzyme-coupled byproduct-release assays, unraveling new mechanistic insights of substrate activation/channeling in NRPS enzymes. PMID:25437123

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

    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. Evolutionary divergence of chloroplast FAD synthetase proteins

    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.

  5. The glutamine synthetase gene family in Populus

    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.

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

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

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

    2016-09-01

    Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA. PMID:27581854

  8. Novel Insights into Regulation of Asparagine Synthetase in Conifers

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

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in ...

  9. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C

  10. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

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

    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...

  12. Prostaglandin synthetase inhibitor in an infant with congenital chloride diarrhoea.

    Minford, A M; Barr, D G

    1980-01-01

    Hyper-reninaemia, hypokaluria, and hypokalaemia in an infant with congenital chloride diarrhoea improved during treatment with a prostaglandin synthetase inhibitor, ketoprofen. There was evidence of increased activity of therenin-aldosterone system when ketoprofen was stopped. It is suggested that prostaglandins may be involved in stimulating the renin-aldosterone system in congenital chloride diarrhoea.

  13. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    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. PMID:25385624

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

    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...

  15. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

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

    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.

  17. Glutamine synthetase gene evolution: A good molecular clock

    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

  18. The glutamine synthetase gene family in Populus

    Cánovas Francisco M; Kirby Edward G; Avila Concepción; Canales Javier; García-Gutiérrez Angel; Castro-Rodríguez Vanessa

    2011-01-01

    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 ...

  19. The aminoacyl-tRNA synthetases of Drosophila melanogaster

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

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translat...

  20. Co-purification of dihydrofolate synthetase and N10formyltetrahydropteroyldiglutamate synthetase from E. coli

    The enzymatic activities which add a single glutamate to dihydropteroate (H2Pte) and to N10formyltetrahydropteroylglutamate (N10formylH4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii . 1.1 microM) of the utilization of N10formylH4PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis . 370 microM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein

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

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

    1984-03-01

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

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

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both [1-14C]-acetate and [214C] malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases. (author)

  3. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin.

    Pascual, María Belén; Jing, Zhong Ping; Kirby, Edward G; Cánovas, Francisco M; Gallardo, Fernando

    2008-01-01

    Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth

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

    El-Waseef, A.

    1982-01-01

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

  5. Holocarboxylase synthetase deficiency pre and post newborn screening

    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.

  6. Holocarboxylase synthetase deficiency pre and post newborn screening.

    Donti, Taraka R; Blackburn, Patrick R; Atwal, Paldeep S

    2016-06-01

    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. PMID:27114915

  7. ß-Lysine discrimination by lysyl-tRNA synthetase

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

    2011-01-01

    Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate a- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site gui...... enantiomers of ß-lysine were substrates for tRNA aminoacylation by LysRS, which, together with the relaxed specificity of the A233S variant, suggest a possible means to develop systems for in vivo co-translational insertion of ß-amino acids.......Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate a- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site...

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

    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 Mg2+ 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.)

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

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

    2008-12-01

    There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 +/- 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations. PMID:19088795

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

    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.)

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

    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.)

  12. Thiol synthetases of legumes: Immunogold localization and differential gene regulation by phytohormones

    Clemente, María Rebeca; Bustos-Sanmamed, Pilar; Loscos Aranda, Jorge; James, Euan Kevin; Pérez-Rontomé, Carmen; Navascués Ortega, Joaquín; Gay, Marina; Becana Ausejo, Manuel

    2012-01-01

    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immu...

  13. Treatment of renal colic by prostaglandin synthetase inhibitors and avafortan (analgesic antispasmodic).

    el-Sherif, A E; Foda, R; Norlen, L J; Yahia, H

    1990-12-01

    In a study of the pain-relieving effect of 3 drugs commonly used to treat acute renal colic in this hospital, intravenous indomethacin and intramuscular diclofenac (prostaglandin synthetase inhibitors) were compared with intravenous Avafortan (analgesic antispasmodic). As first-line analgesics, prostaglandin synthetase inhibitors, if given intravenously, offer an effective alternative to Avafortan. Of 145 patients studied, 32 required a second injection for complete relief of pain. Administering a second dose of prostaglandin synthetase inhibitors resulted in equally significant pain relief rate even though the route was intramuscular. PMID:2265331

  14. Effect of ionizing radiation on methylation of lysil-tRNA-synthetase from rat liver

    X-irradiation of animals with an absolutely lethal dose of 0.21 C/kg increases the incorporation of a methyl group into aminoacyl-tRNA-synthetases the radioactive label being traced in lysine, arginine, and histidine. The isolated protein methyltransferase methylated total preparaions of aminoacyl-tRNA-synthetases and lysil-tRNA-synthetase of rat liver in the in vitro experiments with the use of S-adenosylmethionine-14CH3 as a donor of methyl groups: the radioactive label was traced in arginine, lysine, histidine, aspartic and glytamic acids

  15. Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

    Guo, Li-Tao; Helgadóttir, Sunna; Söll, Dieter; Ling, Jiqiang

    2012-01-01

    Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNAGln is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNAGln, and an amidotransferase converts Glu-tRNAGln to Gln-tRNAGln. The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacyl...

  16. Activity of aminoacyl-tRNA synthetases in experimental hyperthyroidism in muscle tissue of the rabbit

    In cardial and femoral muscles of rabbits specific activities of aminoacyl-tRNA synthetases for twenty amino acids were generally similar, namely the activities towards amino acids and their amides, leucine, isoleucine, histidine, tyrosine, proline and serine were considerably lower than towards the remaining amino acids. Specific activities of most aminoacyl-tRNA synthetases were higher in hyperthyroidism than in euthyreosis, and were higher in femoral muscle than in heart. The response to thyroxine treatment of individual aminoacyl-tRNA synthetases in both kinds of muscles varied with respect to most of the amino acids. (author). 22 refs, 1 fig., 1 tab

  17. In Silico Discovery of Aminoacyl-tRNA Synthetase Inhibitors

    Yaxue Zhao

    2014-01-01

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

  18. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

    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. Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases.

    Iancu, Cristina V; Zhou, Yang; Borza, Tudor; Fromm, Herbert J; Honzatko, Richard B

    2006-09-26

    Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket. PMID:16981730

  20. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Rubin, Edward M.

    1980-01-01

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

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

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

    2006-01-01

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

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

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

    2012-01-01

    Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochon...... aminoacyl-tRNA synthetase as a cause of mitochondrial disease.......Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the...... mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial...

  3. Human tyrosyl-tRNA synthetase shares amino acid sequence homology with a putative cytokine.

    Kleeman, T A; Wei, D; Simpson, K L; First, E A

    1997-05-30

    To test the hypothesis that tRNATyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an amino-terminal Rossmann fold domain that is responsible for formation of the activated E.Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNATyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNATyr, and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyl-tRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of

  4. Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

    Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth; Phillips, Margaret A

    2014-04-01

    Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

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

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

    1985-01-01

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

  6. Oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes in various diseases.

    Fujii, N; Kotake, S.; Hirose, S; Ohno, S; Yasuda, I.; Sagawa, A; Ishikawa, K.; Minagawa, T

    1984-01-01

    Interferon induces oligo-2',5'-adenylate synthetase in cells. In various diseases, interferon was detectable in the circulation or was produced spontaneously from peripheral blood mononuclear leukocytes. The oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes was examined in various diseases, including systemic lupus erythematosus, sarcoidosis, Vogt-Koyanagi-Harada disease, and Behcet's disease. The activity of this enzyme was significantly increased in system...

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

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

    1995-12-10

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

  8. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Das, S.; Gillin, F.D.

    1987-05-01

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

  9. Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii.

    Chen, Q; Silflow, C D

    1996-11-01

    To elucidate the role of glutamine synthetase (GS) in nitrogen assimilation in the green alga Chlamydomonas reinhardtii we used maize GS1 (the cytosolic form) and GS2 (the chloroplastic form) cDNAs as hybridization probes to isolate C. reinhardtii cDNA clones. The amino acid sequences derived from the C. reinhardtii clones have extensive homology with GS enzymes from higher plants. A putative amino-terminal transit peptide encoded by the GS2 cDNA suggests that the protein localizes to the chloroplast. Genomic DNA blot analysis indicated that GS1 is encoded by a single gene, whereas two genomic fragments hybridized to the GS2 cDNA probe. All GS2 cDNA clones corresponded to only one of the two GS2 genomic sequences. We provide evidence that ammonium, nitrate, and light regulate GS transcript accumulation in green algae. Our results indicate that the level of GS1 transcripts is repressed by ammonium but induced by nitrate. The level of GS2 transcripts is not affected by ammonium or nitrate. Expression of both GS1 and GS2 genes is regulated by light, but perhaps through different mechanisms. Unlike in higher plants, no decreased level of GS2 transcripts was detected when cells were grown under conditions that repress photorespiration. Analysis of GS transcript levels in mutants with defects in the nitrate assimilation pathway show that nitrate assimilation and ammonium assimilation are regulated independently. PMID:8938407

  10. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    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 3H 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

  11. Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

    Sun Hui; Huang Qiman; Su Jin

    2005-01-01

    Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.

  12. The aminoacyl-tRNA synthetases of Drosophila melanogaster.

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

    2015-01-01

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

  13. Glutamine synthetase and glutamyltransferase in developing chick and rat tissues

    Herzfeld, A.; Raper, S.M.

    1975-01-01

    Concomitant determination of ..gamma..-glutamine-hydroxylamine-glutamyltransferase (GT) and ..gamma..-glutamyl hydroxamate synthetase (GS) activities in chick retina, brain and liver between the 13th day of incubation and a day after hatching showed that while both activities increased late in incubation, in neural tissues their rises were not simultaneous throughout development; in liver both activities were already high on the 14th day of incubation and changed in parallel thereafter. GS and GT activities could be evoked prematurely in all three tissues by cortisol, but GT activity showed higher responses to the hormone. GT and GS were separable by differential solubilization in chick liver but not in retina of chick or rat. The wide spread of the ratios of GT : GS activities in homogenates of a number of chick and rat tissues (1 : 1 to 73 : 1) indicates that the GS reaction is not catalyzed by the same protein that catalyzes the GT reactions. Relative amounts of GT(T) (free of GS activity) and of variants of GS (with different competences to catalyze the GT reaction) may govern the changes between the two activities during development and their distribution among different adult tissues in chick and rat.

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

    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.

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

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

    2016-08-19

    Nonribosomal peptide synthetases are large, complex multidomain enzymes responsible for the biosynthesis of a wide range of peptidic natural products. Inherent to synthetase chemistry is the thioester templated mechanism that relies on protein/protein interactions and interdomain dynamics. Several questions related to structure and mechanism remain to be addressed, including the incorporation of accessory domains and intermodule interactions. The inclusion of nonproteinogenic d-amino acids into peptide frameworks is a common and important modification for bioactive nonribosomal peptides. Epimerization domains, embedded in nonribosomal peptide synthetases assembly lines, catalyze the l- to d-amino acid conversion. Here we report the structure of the epimerization domain/peptidyl carrier protein didomain construct from the first module of the cyclic peptide antibiotic gramicidin synthetase. Both holo (phosphopantethiene post-translationally modified) and apo structures were determined, each representing catalytically relevant conformations of the two domains. The structures provide insight into domain-domain recognition, substrate delivery during the assembly line process, in addition to the structural organization of homologous condensation domains, canonical players in all synthetase modules. PMID:27294598

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

    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.

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

    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

  18. Characterization of arachidonate 5-lipoxygenase and leukotriene A4 synthetase from RBL-1 cells

    5-lipoxygenase (LO) and leukotriene (LT) A4 synthetase from RBL-1 high speed (105,000 x g for 60 min) supernatants were partially purified by protein-high performance liquid chromatography (HPLC) and characterized in detail. The partially purified preparation contained only 5-LO and LTA4 synthetase and was isolated from 12-LO, peroxidase and LTA4 hydrolase activities. Reaction products were separated by reversed phase HPLC and quantitated by absorption spectrophotometry and radiochemical detection. The enzyme preparation rapidly converted [14C]arachidonate to [14C]5-hydroperoxyeicosatetraenoic acid (HPETE) and [14C]5,12-dihydroperoxyeicosatetraenoic acids (diHETEs). The 5,12-diHETEs were primarily non-enzymatic breakdown products of LTA4 (e.g., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Both the 5-LO and LTA4 synthetase activities were Ca2+- and ATP-dependent. For both enzyme activities, the CA2+ stimulation required the presence of ATP. The fatty acid hydroperoxides, 5-,12-, and 15-HPETE, both stimulated ([ 3 μM]) 5-LO and LTA4 synthetase activities. The rapid isolation and subsequent characterization of 5-LO and LTA4 synthetase provide the bases for the further understanding of the role of the LO pathway in biological processes

  19. Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene

    Jyoti K Jaiswal; Vidyanand Nanjundiah

    2003-12-01

    In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution of GlnRS and AsnRS in eukaryotes.

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

    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...

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

    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...

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

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

    2015-10-20

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

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

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

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...... the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that......, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA...

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

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

    2012-01-01

    In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together...... with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate...... that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs. STRUCTURED SUMMARY OF PROTEIN...

  5. Various mechanisms in cyclopeptide production from precursors synthesized independently of non-ribosomal peptide synthetases

    Wenyan Xu; Liling Li; Liangcheng Du; Ninghua Tan

    2011-01-01

    An increasing number of cyclopeptides have been discovered as products of ribosomal synthetic pathway.The biosynthetic study of these cyclopeptides has revealed interesting new mechanisms for cyclization.This review highlighted the recent discoveries in cyclization mechanisms for cyclopeptides synthesized independently of non-ribosomal peptide synthetases,including endopeptidase-catalyzed cyclization,intein-mediated cyclization,and peptide synthetase-catalyzed cyclization.This information may help to design hybrid ribosomal and non-ribosomal biosynthetic systems to produce novel cyclopeptides with various bioactivities.

  6. A cDNA clone from Zea mays endosperm sucrose synthetase mRNA.

    Geiser, M.; Döring, H P; Wöstemeyer, J; Behrens, U.; Tillmann, E; Starlinger, P.

    1980-01-01

    A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.

  7. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

    Schwer, Bjoern; Bunkenborg, Jakob; Verdin, Regis O; Andersen, Jens S; Verdin, Eric

    2006-01-01

    We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 b...

  8. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera

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

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have not...

  9. The PRPP Synthetase Spectrum: What Does it Demonstrate About Nucleotide Syndromes?

    Duley, J.A.; Christodoulou, J.; Brouwer, A.P.M. de

    2011-01-01

    Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense

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

    2007-01-01

    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.

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

    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

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

    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 guanosin...

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

    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...

  14. Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

    Grevengoed, Trisha J; Martin, Sarah A; Katunga, Lalage; Cooper, Daniel E; Anderson, Ethan J; Murphy, Robert C; Coleman, Rosalind A

    2015-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1(T-/-)) have impaired cardiac fatty acid oxidation and rely on glucose for ATP producti...

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

    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 p

  16. Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

    Revuelta José L

    2008-09-01

    Full Text Available Abstract Background Phosphoribosyl pyrophosphate (PRPP is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability. Results Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C. We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains. Conclusion It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

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

    Byung Sun Park

    2015-01-01

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

  18. Absence of acetohydroxy acid synthetase in a clinical isolate of Neisseria gonorrhoeae requiring isoleucine and valine.

    Lerner, S A; Friedman, E L; Dudek, E J; Kominski, G; Bohnhoff, M.; Morello, J A

    1980-01-01

    A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.

  19. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases.

    Drake, Eric J; Miller, Bradley R; Shi, Ce; Tarrasch, Jeffrey T; Sundlov, Jesse A; Allen, C Leigh; Skiniotis, Georgios; Aldrich, Courtney C; Gulick, Andrew M

    2016-01-14

    Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs). During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases. PMID:26762461

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

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

    1989-01-01

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

  1. An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

    Mazauric, M H; Reinbolt, J; Lorber, B; Ebel, C; Keith, G; Giegé, R; Kern, D

    1996-11-01

    Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict

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

    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.

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

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

    1998-01-01

    . 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......, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded...

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

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

    2012-01-01

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

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

    Hans-Gert eBernstein

    2015-08-01

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

  6. Properties of asparagine synthetase in asparagine-independent variants of Jensen rat sarcoma cells induced by 5-azacytidine.

    Sugiyama, R H; Arfin, S M; Harris, M.

    1983-01-01

    Jensen rat sarcoma cells in culture require L-asparagine for growth and lack detectable levels of asparagine synthetase. Cultures exposed for 24 h to graded concentrations of 5-azacytidine give rise to asparagine-independent variants in high frequency. These prototrophs are stable phenotypically whether maintained in the presence or absence of L-asparagine. Asparagine synthetase activity in several variant clones was uniform in thermolability and several kinetic parameters, as well as in immu...

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

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

    2014-03-11

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

  8. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Sun, Litao; Wagner, Marina; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; Rajbhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela

    2014-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. ...

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

    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...... first report of association between an aminoacyl tRNA synthetase gene and disease. Our results further highlight the important role of mitochondria in glucose homeostasis....

  10. eth-1, the Neurospora crassa locus encoding S-adenosylmethionine synthetase: Molecular cloning, sequence analysis and in vivo overexpression

    Mautino, M.R.; Barra, J.L.; Rosa, A.L. [Universidad Nacional de Cordoba (Argentina)

    1996-03-01

    Intense biochemical and genetic research on the eth-1 mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase). We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1 mutation to clone the locus. Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase. Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme. N. crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression. Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N. crassa even at abnormally high levels of cellular S-Adomet. The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet. We conclude that variation in S-Adomet levels in N. crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis. 90 refs., 5 figs., 1 tab.

  11. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

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

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

    1987-01-01

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

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

    Maha Z. Alriyami

    2014-12-01

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

  14. Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

    A. Wiest; D. Barchers; M. Eaton; R. Henderson; R. Schnittker; K. Mccluskey

    2013-12-01

    Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.

  15. Structure of an unusual S-adenosylmethionine synthetase from Campylobacter jejuni.

    Zano, Stephen P; Pavlovsky, Alexander G; Viola, Ronald E

    2014-02-01

    S-Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group-transfer reactions and also serves as the precursor for two groups of quorum-sensing molecules that function as regulators of the production of virulence factors in Gram-negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacterium Campylobacter jejuni (cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active-site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates. PMID:24531478

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

    O'Hanlon, Karen; Cairns, Timothy; Stack, Deirdre; Schrettl, Markus; Bignell, Elaine; Kavanagh, Kevin; Miggin, Sinead; O'Keeffe, Grainne; Larsen, Thomas; Doyle, Sean

    2011-01-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes...

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

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

    2012-01-01

    Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows ...

  18. Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

    Steinchen, Wieland; Lackner, Gerald; Yasmin, Sabiha; Schrettl, Markus; Dahse, Hans-Martin; Haas, Hubertus; Hoffmeister, Dirk

    2013-01-01

    The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[32P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Usi...

  19. Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

    Fox, Barbara A.; Ristuccia, Jessica G.; Bzik, David J.

    2008-01-01

    New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of UMP and dTMP pools thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CP...

  20. Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

    Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth; Phillips, Margaret A.

    2014-01-01

    Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of...

  1. Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process

    Woese, Carl R.; Olsen, Gary J; Ibba, Michael; Söll, Dieter

    2000-01-01

    The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the followi...

  2. Effect of detergents on membrane-associated glucan synthetase from Paracoccidioides brasiliensis.

    San-Blas, G; San-Blas, F

    1982-01-01

    Yeast and mycelial particulate preparations of Paracoccidioides brasiliensis were subjected to the action of several detergents in an attempt to solubilize the glucan synthetase present in these preparations. This was achieved more successfully in the yeast membranes than in the mycelial ones. The enzymatic activity was greatly stimulated in the insoluble fractions upon treatment with some of the detergents used. The results suggest that the yeast and mycelial phases of P. brasiliensis may di...

  3. Glutamine synthetase immunor present in oligodendroglia of regions of the central nervous system

    D'Amelio, Fernando; Eng, Lawrence F.; Gibbs, Michael A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  4. Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs)

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha; Brouillette, Christie G; Brouillette, Wayne J.

    2009-01-01

    Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies.

  5. Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs)

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha; Brouillette, Christie G.; Brouillette, Wayne J.

    2009-01-01

    Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies. PMID:19249205

  6. Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus⋆

    Gralla, Michael; Camporeale, Gabriela; Zempleni, Janos

    2007-01-01

    The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in respo...

  7. Coexpression of glutamine synthetase and carbamoylphosphate synthase I genes in pancreatic hepatocytes of rat.

    Yeldandi, A. V.; X. D. Tan; Dwivedi, R S; Subbarao, V; Smith, D. D.; Scarpelli, D. G.; Rao, M S; Reddy, J K

    1990-01-01

    In the mammalian liver the distribution of ammonia-detoxifying enzymes, glutamine synthetase (GS) and carbamoylphosphate synthase I (ammonia) (CPS-I), is mutually exclusive in that these enzymes are expressed in two distinct populations of hepatocytes that are zonally demarcated in the liver acinus. In the present study we examined the distribution of GS and CPS-I in pancreatic hepatocytes to ascertain if the expression of these two genes in these hepatocytes is also mutually exclusive. Multi...

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

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

    2007-01-01

    Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechani...

  9. Comparative inhibition of bacterial and microsomal 3-ketodihydrosphingosine synthetases by L-cycloserine and other inhibitors.

    Sundaram, K S; Lev, M

    1984-01-01

    Eleven compounds were examined for their capacity to inhibit the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase. Of these, L-cycloserine was the most potent, affecting both bacterial and brain microsomal enzymes to a significant degree at 0.04 mM. D- and L-cycloserine irreversibly inactivated the enzyme, indicating a suicide substrate mode of action. L-Cycloserine was a more potent inhibitor of the growth of Bacteroides levii than was D-cycloserine, indicating t...

  10. On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.

    C. S. Huang; Moore, W. R.; Meister, A.

    1988-01-01

    gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzyme...

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

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

    2012-01-01

    Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photore...

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

    Carmen M Pérez-Delgado

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

  13. Effect of post-silking drought on nitrogen partitioning and gene expression patterns of glutamine synthetase and asparagine synthetase in two maize (Zea mays L.) varieties.

    Li, Yajun; Wang, Meiling; Zhang, Fengxia; Xu, Yadong; Chen, Xiaohong; Qin, Xiaoliang; Wen, Xiaoxia

    2016-05-01

    Glutamine synthetase (GS) and asparagine synthetase (AS) are proposed to have important function in plant nitrogen (N) remobilization, but their roles under drought stress are not well defined. In this study, the expression dynamics of GS and AS genes were analyzed in two maize varieties (ZD958 and NH101) in relation to post-silking drought stress induced nitrogen partitioning. ZD958 was a 'stay-green' variety with 5% nitrogen harvest index (NHI) lower than NH101. From silking to maturity, the amount of nitrogen remobilized from ear-leaves in ZD958 was evidently lower than NH101, and post-silking drought stress increased the nitrogen remobilization for both varieties. In ear-leaves, the expression of ZmGln1-3 was enhanced under drought stress. Three AS genes (ZmAS1, ZmAS2 and ZmAS3) were differentially regulated by post-silking drought treatment, of which the expression of ZmAS3 was stimulated at late stage of leaf senescence. In NH101, the expression level of ZmAS3 was markedly higher than that in ZD958. In developing grains, there were no significant differences in expression patterns of GS and AS genes between well water and drought treated plants. Drought stress altered maize N partitioning at the whole-plant level, and the up-regulation of GS and AS genes may contribute to the higher leaf nitrogen remobilization when exposed to drought treatments. PMID:26913793

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

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

  15. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    Fice, D; Shen, Z; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism. Images PMID:8384617

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

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

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis. PMID:27234879

  17. Glucosamine synthetase from Escherichia coli: purification, properties and glutamine-utilizing site location

    L-Glutamine: D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction. The enzyme has a K/sub m/ of 2 mM for fructose 6-phosphate, a K/sub m/ of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation

  18. Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

    Onesti, S; Desogus, G; Brevet, A; Chen, J; Plateau, P; Blanquet, S; Brick, P

    2000-10-24

    Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding. PMID:11041850

  19. Structural basis for the binding of succinate to succinyl-CoA synthetase.

    Huang, Ji; Fraser, Marie E

    2016-08-01

    Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate. PMID:27487822

  20. (p)ppGpp synthetases regulate the pathogenesis of zoonotic Streptococcus suis.

    Zhu, Jiawen; Zhang, Tengfei; Su, Zhipeng; Li, Lu; Wang, Dong; Xiao, Ran; Teng, Muye; Tan, Meifang; Zhou, Rui

    2016-10-01

    (p)ppGpp-mediated stringent response is one of the main adaption mechanism in bacteria, and the ability to adapt to environment is linked to the pathogenesis of bacterial pathogens. In the zoonotic pathogen Streptococcus suis, there are two (p)ppGpp synthetases, RelA and RelQ. To investigate the regulatory functions of (p)ppGpp/(p)ppGpp synthetases on the pathogenesis of S. suis, the phenotypes of the [(p)ppGpp(0)] mutant ΔrelAΔrelQ and its parental strain were compared. Light and electron microscopy observation showed that the mutant strain had a longer chain-length than its parental strain. Disruption of relA and relQ led to decreased adhesive and invasive ability to HEp-2 cells, and increased sensitivity to the blood killing and phagocytosis. Mouse infection experiments showed that the mutant strain was attenuated and easier to be cleaned up in vivo. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expressions of virulence related genes involving in morphology and virulence were down-regulated in the mutant strain. Our study demonstrated that the (p)ppGpp synthetases or (p)ppGpp can regulate the pathogenesis of this important zoonotic pathogen. PMID:27524648

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

    Kompanets I. V.; Korotkiy О. G.; Karpovets T. P.; Pilipenko S. V.; Nikolska V. V.; Ostapchenko L. I.; Yankovsky D. S.

    2013-01-01

    The aim of this work was the determination of rat thymocytes response to hypergastrinemia evoked by hypoacidity and multiprobiotic «Symbiter® acidophilic concentrated» (symbiter) treatment via the estimation of the interferon (IFN) titer and 2', 5'-oligoadenylate (OA)-synthetase activity in lymphocytes. 2', 5'-OA-synthetase is the IFN-induced enzyme. Methods. The micromethod of IFN titer determination by antiviral activity, spectrophotometrical method of 2', 5'-OA-synthetase activity determin...

  2. Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

    Kim, Se-Yu; Park, Soo-Young; Choi, Soo-Keun; Park, Seung-Hwan

    2015-07-01

    The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives. PMID:26059516

  3. Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

    Brattsten, L B; Wilkinson, C F

    1975-07-01

    1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase. PMID:1004

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

    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...

  5. REGULATION OF RAT HEPATIC DELTA-AMINOLEVULINIC ACID SYNTHETASE AND HEME OXYGENASE ACTIVITIES: EVIDENCE FOR CONTROL BY HEME AND AGAINST MEDIATION BY PROSTHETIC IRON

    The effects of in vivo administration of 6 compounds on the activity of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. The order of decreasing potency in reducing ALA synthetase activity was heme, bilirubin, protoporphyrin IX, bilirubin dimethyl es...

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

    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

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

    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 L-

  8. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

    2016-01-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  9. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Mertens, Haydyn; Svergun, Dmitri; Brieba, Luis G; Grøtli, Morten; Torres-Larios, Alfredo

    2016-07-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  10. Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn.

    Jiang, Enshe; Yan, Xisheng; Weng, Han-Rong

    2012-05-01

    Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions. PMID:22339645

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

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

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. PMID:26205258

  12. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K2) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C2221, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

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

    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. PMID:27125317

  14. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    Fice, D; Z. Shen; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the ...

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

    Ray, P H

    1980-01-01

    3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to b...

  16. A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

    Arriarán, Sofía; Agnelli, Silvia; Fernández López, José Antonio; Remesar Betlloch, Xavier; Alemany, Marià

    2012-01-01

    A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium w...

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

    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...... vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA(Glu) was able to protect GluRS1 against oxidative...

  18. Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

    Rudinger, J; Puglisi, J D; Pütz, J.; Schatz, D; Eckstein, F; Florentz, C; Giegé, R

    1992-01-01

    The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of...

  19. Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp.

    Fuchs, R L; Keister, D L

    1980-01-01

    Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium....

  20. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF†

    Francisco, Brian San; Bretsnyder, Eric C.; Rodgers, Kenton R.; Kranz, Robert G.

    2011-01-01

    Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conser...

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

    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.

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

    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

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

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman;

    2004-01-01

    -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.......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...

  4. Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

    Yao, Peng; Zhu, Bin; Jaeger, Sophie; Eriani, Gilbert; Wang, En-Duo

    2008-01-01

    Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNALeu recognition sets and their evolution, the recognition of tRNALeu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and ...

  5. Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes*

    Lobo, Sandra; Wiczer, Brian M.; Bernlohr, David A

    2009-01-01

    ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptak...

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

    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 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....

  7. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  8. Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation.

    Brevet, Annie; Chen, Josiane; Commans, Stéphane; Lazennec, Christine; Blanquet, Sylvain; Plateau, Pierre

    2003-08-15

    The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases. PMID:12766171

  9. Crystallization and preliminary crystallographic characterization of glutamine synthetase from Medicago truncatula

    The enzyme glutamine synthetase from M. truncatula has been expressed, purified and crystallized. The crystals belonged to the monoclinic space group P21 and diffracted to 2.35 Å resolution. The condensation of ammonium and glutamate into glutamine catalyzed by glutamine synthetase (GS) is a fundamental step in nitrogen metabolism in all kingdoms of life. In plants, this is preceded by the reduction of inorganic nitrogen to an ammonium ion and therefore effectively articulates nitrogen fixation and metabolism. Although the three-dimensional structure of the dodecameric bacterial GS was determined quite some time ago, the quaternary architecture of the plant enzyme has long been assumed to be octameric, mostly on the basis of low-resolution electron-microscopy studies. Recently, the crystallographic structure of a monocotyledonous plant GS was reported that revealed a homodecameric organization. In order to unambiguously establish the quaternary architecture of GS from dicotyledonous plants, GS1a from the model legume Medicago truncatula was overexpressed, purified and crystallized. The collection of synchrotron diffraction data to 2.35 Å resolution allowed the determination of the three-dimensional structure of this enzyme by molecular replacement

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

    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. PMID:27225077

  11. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

    Novoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marín-García, Patricia; Azcárate, Isabel G; Bautista, José M; Mirando, Adam C; Francklyn, Christopher S; Varon, Sònia; Royo, Miriam; Cortés, Alfred; Ribas de Pouplana, Lluís

    2014-12-23

    Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo. PMID:25489076

  12. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  13. Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

    Reimer, Janice M; Aloise, Martin N; Harrison, Paul M; Schmeing, T Martin

    2016-01-14

    Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics. PMID:26762462

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

    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.

  15. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

    Koryakina, Irina; McArthur, John; Randall, Shan; Draelos, Matthew M; Musiol, Ewa M; Muddiman, David C; Weber, Tilmann; Williams, Gavin J

    2013-01-18

    Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides. PMID:23083014

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

    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.

  17. Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)

    The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), [2-3H]8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of [2-3H]-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-[14C]glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure

  18. CcsBA is a cytochrome c synthetase that also functions in heme transport

    Frawley, Elaine R.; Kranz, Robert G.

    2009-01-01

    Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel. PMID:19509336

  19. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

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

    Kumada, Yoichi; Takano, Eriko; Nagaoka, Kozo

    1990-01-01

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

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

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

    2006-01-01

    an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was...

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

    Thykjaer, T; Danielsen, D; She, Q;

    1997-01-01

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

  3. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

    Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

    1988-01-01

    The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to...

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

    Martinussen, Jan; Hammer, Karin

    1998-01-01

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

  5. Glucosamine synthetase from Escherichia coli: kinetic mechanism and inhibition by N3-fumaroyl-L-2,3-diaminopropionic derivatives

    N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropionic acid (FMDP; 1, R = OMe), a member of a new class of glutamine analogues, has been investigated as an inhibitor of pure Escherichia coli glucosamine synthetase. Product and dead-end inhibition studies indicate an ordered association to the enzyme with the sugar molecule binding prior to substrate or inhibitor. The inactivation exhibits pseudo-first-order kinetics, is irreversible, and occurs faster in the presence of fructose 6-phosphate, a behavior previously reported for the partially purified enzyme from Salmonella typhimurium. Inhibition occurs with partial covalent incorporation of L-FMDP into glucosamine synthetase. In the presence of fructose 6-phosphate, enzyme inactivation with [2-3H]-DL-FMDP is associated with the incorporation of 0.75 equiv of inhibitor and with the modification of 0.78 thiol residue per enzyme subunit. This result is the first evidence for covalent entrapment of the entire inhibitor molecule following FMDP-mediated glucosamine synthetase inactivation. Preliminary inactivation with 6-diazo-5-oxo-L-norleucine, known to alkylate selectively the NH2-terminal cysteine residue, completely prevents radioactivity incorporation. Therefore, this inhibitor is postulated to covalently modify glucosamine synthetase through direct addition of the thiol nucleophile from the terminal cysteine residue to the Michael acceptor 1, so acting as an affinity label rather than a mechanism-based inhibitor

  6. Phenotypic Changes Resulting from Distinct Point Mutations in the Azospirillum brasilense glnA Gene, Encoding Glutamine Synthetase

    Van Dommelen, Anne; Keijers, Veerle; Wollebrants, An; Vanderleyden, Jozef

    2003-01-01

    Sequencing the glnA genes of two chemically induced Azospirillum brasilense glutamine synthetase mutants revealed an Arg→Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp→Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.

  7. Radiosensitivity of glutathione-synthetase deficient human fibroblasts in vitro: Indication of a correlation between OER and glutathione-synthetase activity

    5-oxoprolinuria is an autosomal recessive disease associated with a glutathione-synthetase (GSH-S) deficiency. Previous results obtained with a fibroplast cell strain originating from such a patient (VP) and exhibiting a decreased glutathione (GSH) level, showed a reduced oxygen enhancement ratio (OER) (1.5; control: 2.9). In order to assess the radioprotective role of the intrinsic GSH, radiosensitivity of 6 fibroblast cell strains with different GSH-S activity have been studied using colony assay as endpoint. 3 strains originated from 5-oxoprolinuria patients (OB, EB and AZ); 3 other strains were got from parents of patients (RB, JB and SP). Among those 6 cell strains, the GSH level was found to be depleted only in AZ and SP. However, a reduced OER is observed for the 6 cell strains (1.7, 2.0 and 2.0 for the patients; 1.9, 2.2 and 2.7 for parents; controls: 2.9). As far as survival of GSH-S deficient cells is concerned, OER seems to be correlated with GSH-S activity: the lower the GSH-S activity, the lower the OER. These results are discussed taking into account other unpublished results obtained from experiments designed to investigate the role of GSH in radio-induced enzymatic repair

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

    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

  9. Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

    Finke, A.; Roberts, I.; Boulnois, G; Pzzani, C; Jann, K

    1989-01-01

    The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of th...

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

    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...... 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...

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

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

    2012-06-01

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

  12. Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

    Liliana Torcoroma García

    2007-09-01

    Full Text Available The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

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

    The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene

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

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

    2009-01-01

    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...... 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 OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS...

  15. Radiosensitizing and cytotoxic properties of misonidazole on glutathione synthetase deficient human fibroblasts

    The cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0.1 to 4 mM, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strains concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1.5 and 1.5 for deficient cells, 2.8 and 2.9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells. (author)

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

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

    2014-01-01

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

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

    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.

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

    Langston-Unkefer, P.J.

    1991-06-11

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

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

    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...... contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P <0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of...... metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus...

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

    Marco Betti

    2012-06-01

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

  1. Peculiarities and impacts of expression of bacterial cyanophycin synthetases in plants.

    Nausch, Henrik; Huckauf, Jana; Broer, Inge

    2016-02-01

    Cyanophycin (CP) can be successfully produced in plants by the ectopic expression of the CphA synthetase from Thermosynechococcus elongatus BP-1 (Berg et al. 2000), yielding up to 6.8 % of dry weight (DW) in tobacco leaf tissue and 7.5 % in potato tubers (Huehns et al. 2008, 2009). Though, high amounts of the polymer lead to phenotypical abnormalities in both crops. The extension of abnormalities and the maximum amount of CP tolerated depend on the compartment that CP production is localized at the tissue/crop in which CP was produced (Huehns et al. 2008, 2009; Neumann et al. 2005). It cannot be ascribed to a depletion of arginine, lysine, or aspartate, the substrates for CP synthesis. PMID:26658983

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

    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.

  3. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF†

    Francisco, Brian San; Bretsnyder, Eric C.; Rodgers, Kenton R.; Kranz, Robert G.

    2011-01-01

    Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a “cavity” is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (Em,7 = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase. PMID:22066495

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

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

    1983-01-01

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

  5. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

    Hove-Jensen, Bjarne

    1985-01-01

    The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage λD69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by...... lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent...... deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the...

  6. The Populus Superoxide Dismutase Gene Family and Its Responses to Drought Stress in Transgenic Poplar Overexpressing a Pine Cytosolic Glutamine Synthetase (GS1a)

    Molina-Rueda, Juan Jesús; Tsai, Chung Jui; Kirby, Edward G.

    2013-01-01

    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...

  7. Nostophycin Biosynthesis Is Directed by a Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase in the Toxic Cyanobacterium Nostoc sp. Strain 152▿†

    Fewer, David P.; Österholm, Julia; Rouhiainen, Leo; Jokela, Jouni; Wahlsten, Matti; Sivonen, Kaarina

    2011-01-01

    Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of th...

  8. Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

    Trauger, John W.; Walsh, Christopher T.

    2000-01-01

    The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step to...

  9. The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

    Durbecq, Virginie; Legrain, Christianne; Roovers, Martine; Piérard, André; Glansdorff, Nicolas

    1997-01-01

    Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found...

  10. Improvement of an l-Leucine-Producing Mutant of Brevibacterium lactofermentum 2256 by Genetically Desensitizing It to α-Acetohydroxy Acid Synthetase

    Tsuchida, Takayasu; Momose,Haruo

    1986-01-01

    Genetic improvement of l-leucine productivity in strain 218, an ile− 2-thiazolealanine-resistant mutant of Brevibacterium lactofermentum 2256, was attempted. In strain 218, which produced 28 mg of l-leucine per ml from 13% glucose, α-isopropylmalate synthetase was genetically desensitized and derepressed to the effect of l-leucine, whereas α-acetohydroxy acid synthetase remained unaltered, although it could be derepressed phenotypically by limiting the isoleucine concentration in the culture....

  11. The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE

    Francisco, Brian San; Sutherland, Molly C.; Kranz, Robert G.

    2014-01-01

    Cytochrome c maturation (ccm) in many bacteria, archaea, and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches heme covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of heme to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation ...

  12. Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining t...

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

    Kompanets I. V.

    2013-01-01

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

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

    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

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

    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

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

    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

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

    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.

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

    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...

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

    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...

  20. Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture

    Neuenfeldt, Anne; Lorber, Bernard; Ennifar, Eric; Gaudry, Agnès; Sauter, Claude; Sissler, Marie; Florentz, Catherine

    2012-01-01

    In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS)...

  1. A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

    Cestari, Igor; Kalidas, Savitha; Monnerat, Severine; Anupama, Atashi; Phillips, Margaret A.; Stuart, Kenneth

    2013-01-01

    The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA syn...

  2. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathoge...

  3. Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis†

    Lu, Xuequan; Zhang, Huaning; Tonge, Peter J.; Tan, Derek S.

    2008-01-01

    Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an ...

  4. Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria

    Kaczmarzyk, Danuta; Hudson, Elton P.; Fulda, Martin

    2016-01-01

    Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty ...

  5. Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

    Leaphart, A. B.; Friez, M. J.; Lovell, C R

    2003-01-01

    Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.

  6. The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB g...

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

    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...

  8. Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.

    Han, B D; Nolan, W G; Hopkins, H P; Jones, R. T.; Ingraham, J L; Abdelal, A T

    1990-01-01

    The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of tempe...

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

    Rongzhong Li

    2015-07-01

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

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

    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.

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

    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 P6122 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 P6122 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. Splicing Defect in Mitochondrial Seryl-tRNA Synthetase Gene Causes Progressive Spastic Paresis Instead of HUPRA Syndrome.

    Linnankivi, Tarja; Neupane, Nirajan; Richter, Uwe; Isohanni, Pirjo; Tyynismaa, Henna

    2016-09-01

    Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function. PMID:27279129

  13. On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes.

    Fisher, M T

    1993-07-01

    For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions. Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T. (1992) Biochemistry 31, 3955-3963). In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins. When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline. The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent. It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate. If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly. PMID:8100224

  14. Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

    Fox, Barbara A; Ristuccia, Jessica G; Bzik, David J

    2009-04-01

    New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development. PMID:18992249

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

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

    2011-12-01

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

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

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

    2015-01-01

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

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

    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.

  18. Intestinal acyl-CoA synthetase 5: Activation of long chain fatty acids and behind

    Christina Klaus

    2013-01-01

    Full Text Available The intestinal mucosa is characterized by a high complexity in terms of structure and functions and allows for a controlled demarcation towards the gut lumen. On the one hand it is responsible for pulping and selective absorption of alimentary substances ensuring the immunological tolerance, on the other hand it prevents the penetration of micro-organisms as well as bacterial outgrowth. The continuous regeneration of surface epithelia along the crypt-villus-axis in the small intestine is crucial to assuring these various functions. The core phenomena of intestinal epithelia regeneration comprise cell proliferation, migration, differentiation, and apoptosis. These partly contrarily oriented processes are molecularly balanced through numerous interacting signaling pathways like Wnt/β-catenin, Notch and Hedgehog, and regulated by various modifying factors. One of these modifiers is acyl-CoA synthetase 5 (ACSL5. It plays a key role in de novo lipid synthesis, fatty acid degradation and membrane modifications, and regulates several intestinal processes, primarily through different variants of protein lipidation, e.g., palmitoylation. ACSL5 was shown to interact with proapoptotic molecules, and besides seems to inhibit proliferation along the crypt-villus-axis. Because of its proapoptotic and antiproliferative characteristics it could be of significant relevance for intestinal homeostasis, cellular disorder and tumor development.

  19. Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro.

    Zheng, Sheng; Zhu, Dan; Lian, Xue; Liu, Weiting; Cao, Ruibing; Chen, Puyan

    2016-05-01

    The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study. PMID:26437676

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

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

    2016-01-01

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

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

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

    2016-01-01

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

  2. An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

    SUNJIANGE; XIAOLIYAO; 等

    1998-01-01

    In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

  3. A plant malonyl-CoA synthetase enhances lipid content and polyketide yield in yeast cells.

    Wang, Yechun; Chen, Hui; Yu, Oliver

    2014-06-01

    Malonyl-CoA is the essential building block of natural products such as fatty acids, polyketides, and flavonoids. Engineering the biosynthesis of fatty acids is important for biofuel production while that of polyketides provides precursors of medicines and nutritional supplements. However, microorganisms maintain a small amount of cellular malonyl-CoA, which could limit production of lipid and polyketides under certain conditions. Malonyl-CoA concentration is regulated by multiple pathways and signals, and changes in intracellular malonyl-CoA often lead to complex alterations in metabolism. In the present work, overexpression of a plant malonyl-CoA synthetase gene (AAE13) in Saccharomyces cerevisiae resulted in 1.6- and 2.4-fold increases in lipid and resveratrol accumulation simultaneously. We also demonstrated that AAE13 partially complemented the temperature-sensitive acc1 mutant, replacing this key enzyme in central metabolism. Mechanistic analysis by CoA quantification and transcriptomic measurement suggested that increases in malonyl-CoA concentration were coupled with drastic reductions in other major CoA compounds and clear suppression of tricarboxylic acid cycle-related genes. These results suggest that malonyl-CoA is a critical target for fatty acid and polyketide engineering and that overexpression of malonyl-CoA synthetic enzymes needs to be combined with upregulation of CoA synthesis to maintain metastasis of central metabolism. PMID:24682482

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

    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.

  5. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

    Madiraju, Anila K; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2016-06-14

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

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

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

    2015-09-01

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

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

    Selenophosphate synthetase 1 (SPS1) is an essential cellular gene in higher eukaryotes. Five alternative splice variants of human SPS1 (major type, ΔE2, ΔE8, +E9, +E9a) were identified wherein +E9 and +E9a make the same protein. The major type was localized in both the nuclear and plasma membranes, and the others in the cytoplasm. All variants form homodimers, and in addition, the major type forms a heterodimer with ΔE2, and ΔE8 with +E9. The level of expression of each splice variant was different in various cell lines. The expression of each alternative splice variant was regulated during the cell cycle. The levels of the major type and ΔE8 were gradually increased until G2/M phase and then gradually decreased. ΔE2 expression peaked at mid-S phase and then gradually decreased. However, +E9/+E9a expression decreased gradually after cell cycle arrest. The possible involvement of SPS1 splice variants in cell cycle regulation is discussed.

  8. Nonribosomal peptide synthetase with a unique iterative-alternative-optional mechanism catalyzes amonabactin synthesis in Aeromonas.

    Esmaeel, Qassim; Chevalier, Mickael; Chataigné, Gabrielle; Subashkumar, Rathinasamy; Jacques, Philippe; Leclère, Valérie

    2016-10-01

    Based on the exploration of data generated by genome sequencing, a bioinformatics approach has been chosen to identify the biosynthetic pathway of the siderophores produced by Aeromonas species. The amonabactins, considered as a virulence factor, represent a family of four variants of catechol peptidic siderophores containing Dhb, Lys, Gly, and an aromatic residue either Trp or Phe in a D-configuration. The synthesis operon is constituted of seven genes named amoCEBFAGH and is iron-regulated. The cluster includes genes encoding proteins involved in the synthesis and incorporation of the Dhb monomer, and genes encoding specific nonribosomal peptide synthetases, which are responsible for the building of the peptidic moiety. The amonabactin assembly line displays a still so far not described atypical mode of synthesis that is iterative, alternative, and optional. A disruption mutant in the adenylation domain of AmoG was unable to synthesize any amonabactin and to grow in iron stress conditions while a deletion of amoH resulted in the production of only two over the four forms. The amo cluster is widespread among most of the Aeromonas species, only few species produces the enterobactin siderophore. PMID:27531515

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

    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.

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

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

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

    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.

  12. Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum.

    Pasaje, Charisse Flerida A; Cheung, Vanessa; Kennedy, Kit; Lim, Erin E; Baell, Jonathan B; Griffin, Michael D W; Ralph, Stuart A

    2016-01-01

    The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNA(trp). Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNA(trp) while the cytosolic TrpRS charges eukaryotic tRNA(trp). An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target. PMID:27277538

  13. Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene.

    Wang, Yong; Boghigian, Brett A; Pfeifer, Blaine A

    2007-11-01

    An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l(-1) OD(600)(-1). In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds. PMID:17876579

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

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

    2016-01-01

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

  15. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    Gabriela Beatriz Acosta; María Alejandra Fernández; Diego Martín Roselló; María Luján Tomaro; Karina Balestrasse; Abraham Lemberg

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into shamoperated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions.

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

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl2, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

  17. In vitro characterization of the NAD(+) synthetase NadE1 from Herbaspirillum seropedicae.

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

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. PMID:26802007

  18. Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia.

    Hsiu-Ni Kung

    2011-08-01

    Full Text Available Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS. Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.

  19. Main: 1W07 [RPSD[Archive

    Full Text Available 1; Arabidopsis Thaliana Molecule: Acyl-Coa Oxidase; Chain: A, B; Mutation: Yes; Engineered: Yes Oxidoreducta...AGSRHAFEVSDRIARLVASDPVFEKSNRARLSRKELFKSTLRKCAHAFKRIIELRLNEEEAGRLRHFIDQPAYVDLHWGMFVPAI...YLWCSGLPELFAVYVPACTYEGDNVVLQLQVARFLMKTVAQLGSGKVPVGTTAYMGRAAHLLQCRSGVQKAEDWLNPDVVLEAFEARALRMAVTCAKNLSKFENQEQGFQELLADLVEAAI

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

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

    2012-03-26

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

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

    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

  2. The Roles of Compensatory Evolution and Constraint in Aminoacyl tRNA Synthetase Evolution.

    Adrion, Jeffrey R; White, P Signe; Montooth, Kristi L

    2016-01-01

    Mitochondrial protein translation requires interactions between transfer RNAs encoded by the mitochondrial genome (mt-tRNAs) and mitochondrial aminoacyl tRNA synthetase proteins (mt-aaRS) encoded by the nuclear genome. It has been argued that animal mt-tRNAs have higher deleterious substitution rates relative to their nuclear-encoded counterparts, the cytoplasmic tRNAs (cyt-tRNAs). This dynamic predicts elevated rates of compensatory evolution of mt-aaRS that interact with mt-tRNAs, relative to aaRS that interact with cyt-tRNAs (cyt-aaRS). We find that mt-aaRS do evolve at significantly higher rates (exemplified by higher dN and dN/dS) relative to cyt-aaRS, across mammals, birds, and Drosophila. While this pattern supports a model of compensatory evolution, the level at which a gene is expressed is a more general predictor of protein evolutionary rate. We find that gene expression level explains 10-56% of the variance in aaRS dN/dS, and that cyt-aaRS are more highly expressed in addition to having lower dN/dS values relative to mt-aaRS, consistent with more highly expressed genes being more evolutionarily constrained. Furthermore, we find no evidence of positive selection acting on either class of aaRS protein, as would be expected under a model of compensatory evolution. Nevertheless, the signature of faster mt-aaRS evolution persists in mammalian, but not bird or Drosophila, lineages after controlling for gene expression, suggesting some additional effect of compensatory evolution for mammalian mt-aaRS. We conclude that gene expression is the strongest factor governing differential amino acid substitution rates in proteins interacting with mitochondrial versus cytoplasmic factors, with important differences in mt-aaRS molecular evolution among taxonomic groups. PMID:26416980

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

    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.

  4. A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum

    Santoni Daniele

    2009-12-01

    Full Text Available Abstract Background Plasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. Results Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv several PfaaRSs posses very unusual domain architectures; (v phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. Conclusion We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

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

    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

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

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of α-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO43- allosteric site where PO43- functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

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

    Uly A. Nikmah

    2016-04-01

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

  8. Pharmaceutical inhibition of glycogen synthetase kinase 3 beta suppresses wear debris-induced osteolysis.

    Geng, Dechun; Wu, Jian; Shao, Hongguo; Zhu, Shijun; Wang, Yijun; Zhang, Wen; Ping, Zichuan; Hu, Xuanyang; Zhu, Xuesong; Xu, Yaozeng; Yang, Huilin

    2015-11-01

    Aseptic loosening is associated with the development of wear debris-induced peri-implant osteolytic bone disease caused by an increased osteoclastic bone resorption and decreased osteoblastic bone formation. However, no effective measures for the prevention and treatment of peri-implant osteolysis currently exist. The aim of this study was to determine whether lithium chloride (LiCl), a selective inhibitor of glycogen synthetase kinase 3 beta (GSK-3β), mitigates wear debris-induced osteolysis in a murine calvarial model of osteolysis. GSK-3β is activated by titanium (Ti) particles, and implantation of Ti particles on the calvarial surface in C57BL/6 mice resulted in osteolysis caused by an increase in the number of osteoclasts and a decrease in the number of osteoblasts. Mice implanted with Ti particles were gavage-fed LiCl (50 or 200 mg kg(-1)d(-1)), 6 days per week for 2 weeks. The LiCl treatment significantly inhibited GSK-3β activity and increased β-catenin and axin-2 expression in a dose-dependent manner, dramatically mitigating the Ti particle-induced suppression of osteoblast numbers and the expression of bone formation markers. Finally, we demonstrated that inhibition of GSK-3β suppresses osteoclast differentiation and reduces the severity of Ti particle-induced osteolysis. The results of this study indicate that Ti particle-induced osteolysis is partly dependent on GSK-3β and, therefore, the canonical Wnt signaling pathway. This suggests that selective inhibitors of GSK-3β such as LiCl may help prevent and treat wear debris-induced osteolysis. PMID:26275858

  9. Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based.

    Williams, Tishan L; Yin, Yuhui W; Carter, Charles W

    2016-01-01

    Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS. PMID:26555258

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

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

    2013-06-01

    Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance. PMID:23542183

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

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

    2015-03-15

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

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

    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.

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

    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

  14. The effect of gamma irradiation on astaxanthin synthetase encoding gene in two mutant strains of Phaffia rhodozyma.

    Naeimeh Najafi

    2013-09-01

    Full Text Available Astaxanthin, an orange-red carotenoid pigment, acts as a protective agent against oxidative damage to cells in vivo. The astaxanthin synthetase gene (crtS size consists of 3995 bp. This gene has been suggested to catalyse β-carotene to astaxanthin in Phaffia rhodozyma. The aim of this research was to find any possible changes in this gene in two mutant strains, Gam1 and Gam2 (with high astaxanthin pigment production, previously created by gamma irradiation.The astaxanthin synthetase gene sequence of Phaffia rhodozyma in the NCBI Gene bank was used to design primer. In Gam1, this gene was amplified using primers Asta F1, Asta R2, Asta F3, Asta R4. In Gam2, primers asta F1, asta R4 were used to amplify the gene. The amplified fragments were 8 sequenced using primers Asta F1, Asta R1, Asta F2, Asta R2, Asta F3, Asta R3 and Asta F4, Asta R4. Astaxanthin synthetase gene from two mutant strains, Gam1 and Gam2 were amplified using PCR. The amplified products were sequenced and aligned using the ClustalW software.The comparison of this gene showed 98% and 99% similarities between the reference sequence and Gam1 and Gam2 mutant strains, respectively, whereas the comparison of this gene in Gam1 and Gam2 mutant strains showed 97% similarity. However, the deduced proteins showed 78% and 83% between the reference protein obtained from the wild type and Gam1 and Gam2, respectively. This similarity was 75% between the mutant strains.

  15. Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea.

    Ostrowski, Maciej; Mierek-Adamska, Agnieszka; Porowińska, Dorota; Goc, Anna; Jakubowska, Anna

    2016-10-01

    Phytohormone conjugation is one of the mechanisms that maintains a proper hormonal homeostasis and that is necessary for the realization of physiological responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases convert indole-3-acetic acid (IAA) to IAA-amino acid conjugates by ATP-dependent reactions. IAA-aspartate (IAA-Asp) exists as a predominant amide conjugate of auxin in pea tissues and acts as an intermediate during IAA catabolism. Here we report a novel recombinant indole-3-acetic acid-amido synthetase in Pisum sativum. In silico analysis shows that amino acid sequence of PsGH3 has the highest homology to Medicago truncatula GH3.3. The recombinant His-tag-PsGH3 fusion protein has been obtained in E. coli cells and is a soluble monomeric polypeptide with molecular mass of 69.18 kDa. The PsGH3 was purified using Ni(2+)-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing Km(ATP) = 0.49 mM, Km(L-Asp) = 2.2 mM, and Km(IAA) = 0.28 mM. Diadenosine pentaphosphate (Ap5A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating Ki = 8.5 μM. L-Tryptophan acts as an inhibitor of IAA-amido synthesizing activity by competition with L-aspartate. Inorganic pyrophosphatase (PPase) hydrolyzing pyrophosphate to two phosphate ions, potentiates IAA-Asp synthetase activity of PsGH3. Our results demonstrate that PsGH3 is a novel enzyme that is involved in auxin metabolism in pea seeds. PMID:27235647

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

    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...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

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

    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 in a...... DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up to...... nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

  18. Gamma-irradiation effect on the acceptor activity of phenylalanine-tRNA and aminoacyl-tRNA-synthetases of cotton seeds

    Changes in the activity of phenylalanine-tRNA-synthetases in the reaction of addition of C14-phenylalanine to tRNA, as well as in the acceptor activity of phenylalanine-tRNA itself and the substrate specificity of phenylalanine--tRNA-synthetases have been studied in two-day-old cotton seedlings grown from seeds irradiated with doses of 6, 25 and 50 kR. Phenylalanine-tRNA and its synthetase, and the isoacceptor tRNAs were shown to be differently sensitive to the action of ionizing radiation. The activity of enzyme was inhibited more markedly than the corresponding tRNA. Study of the substrate specificity of each fraction showed that enzyme E1 acylated isoacceptor phenylalanine-tRNA 1 and 2; the enzyme E2, isoacceptor form 3

  19. Chemical modification of the tryptophan residues of leucyl-tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide

    The accessibility for modification of the tryptophan residues in leucyl-tRNA synthetase from cow mammary glands has been studied with the aid of the specific chemical reagents N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. UV absorption and the intrinsic fluorescence of the tryptophan residues of the enzymes were used for recording the course of the modification. It has been shown that under native conditions (pH 7.8) two superficial residues in each subunit of the dimeric enzyme undergo modification. Under denaturing conditions (6 M guanidine hydrochloride), the internal tryptophan residues are also modified. When the tryptophan residues are modified, the leucyl-tRNA synthetase is inactivated both in the aminoacylation reaction and in the ATP-PP/sub i/ exchange reaction. In the specific complex of leucyl-tRNA synthetase with tRNA/sup Leu/ one of the superficial tryptophan residues is screened and does not undergo modification by the reagents used

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

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

    1999-01-01

    Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-...

  1. Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids

    Ford, Tyler J.; Way, Jeffrey C

    2015-01-01

    FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6–12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reve...

  2. Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

    Kwon, O; Bhattacharyya, D K; Meganathan, R

    1996-01-01

    The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as dete...

  3. The Synechococcus strain PCC 7942 glnN product (glutamine synthetase III) helps recovery from prolonged nitrogen chlorosis.

    Sauer, J; Dirmeier, U; Forchhammer, K

    2000-10-01

    We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacterium Synechococcus strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of glnN is impaired in NtcA- and P(II)-deficient mutants. The only parameter which was negatively affected in the glnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen. PMID:10986271

  4. The Synechococcus Strain PCC 7942 glnN Product (Glutamine Synthetase III) Helps Recovery from Prolonged Nitrogen Chlorosis

    Sauer, Jörg; Dirmeier, Ulrike; Forchhammer, Karl

    2000-01-01

    We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacterium Synechococcus strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of glnN is impaired in NtcA- and PII-deficient mutants. The only parameter which was negatively affected in the glnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-star...

  5. A Nonribosomal Peptide Synthetase with a Novel Domain Organization Is Essential for Siderophore Biosynthesis in Vibrio anguillarum

    Di Lorenzo, Manuela; Poppelaars, Sophie; Stork, Michiel; Nagasawa, Maho; Tolmasky, Marcelo E.; Crosa, Jorge H.

    2004-01-01

    Anguibactin, a siderophore produced by Vibrio anguillarum, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. We have identified a gene from the V. anguillarum plasmid pJM1 that encodes a 78-kDa NRPS protein termed AngM, which is essential in the biosynthesis of anguibactin. The predicted AngM amino acid sequence shows regions of homology to the consensus sequence for the peptidyl carrier protein (PCP) and the condensation (C) domains of NRPSs, and curiously, these two dom...

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

    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.

  7. Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

    Miller Andrew D

    2003-06-01

    Full Text Available Abstract Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common

  8. Highly variable clinical phenotype of carbamylphosphate synthetase 1 deficiency in one family: an effect of allelic variation in gene expression?

    Klaus, V; Vermeulen, T; Minassian, B;

    2009-01-01

    Deficiency of the urea cycle enzyme carbamylphosphate synthetase 1 (CPS1) causes hyperammonemia with a vast range of clinical severity from neonatal onset with early lethality to onset after age 40 with rare episodes of hyperammonemic confusion. The cause for this variability is not understood. We...... report two patients from one family with highly divergent clinical course, one presenting neonatally with a fatal form and the other at age 45 with benign diet-responsive disease. The patients are compound heterozygous for two mutations of the CPS1 gene, c.3558 + 1G > C and c.4101 + 2T > C...

  9. Inhibition of Methionyl-tRNA Synthetase by REP8839 and Effects of Resistance Mutations on Enzyme Activity▿

    Green, Louis S.; Bullard, James M; Ribble, Wendy; Dean, Frank; Ayers, David F.; Ochsner, Urs A.; Janjic, Nebojsa; Jarvis, Thale C.

    2008-01-01

    REP8839 is a selective inhibitor of methionyl-tRNA synthetase (MetRS) with antibacterial activity against a variety of gram-positive organisms. We determined REP8839 potency against Staphylococcus aureus MetRS and assessed its selectivity for bacterial versus human orthologs of MetRS. The inhibition constant (Ki) of REP8839 was 10 pM for Staphylococcus aureus MetRS. Inhibition of MetRS by REP8839 was competitive with methionine and uncompetitive with ATP. Thus, high physiological ATP levels w...

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

    Anderson, John S.; Alexander, Alice A.; Lingyi Lynn Deng; Sijin Lei

    2010-01-01

    The cell wall teichuronic acid (TUA) of Micrococcus luteus is a long-chain polysaccharide composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6)α-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...

  11. Elucidating the Pseudomonas aeruginosa Fatty Acid Degradation Pathway: Identification of Additional Fatty Acyl-CoA Synthetase Homologues

    Zarzycki-Siek, Jan; Norris, Michael H.; Kang, Yun (Kenneth); Sun, Zhenxin; Bluhm, Andrew P.; McMillan, Ian A.; Hoang, Tung T.

    2013-01-01

    The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. T...

  12. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.;

    1989-01-01

    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 by chemi...... cation binds to PRPP synthetase and serves as a bridge to the α-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site....

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

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

    2007-05-01

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

  14. A multiple aminoacyl-tRNA synthetase complex that enhances tRNA-aminoacylation in African trypanosomes.

    Cestari, Igor; Kalidas, Savitha; Monnerat, Severine; Anupama, Atashi; Phillips, Margaret A; Stuart, Kenneth

    2013-12-01

    The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development. PMID:24126051

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

    Willibald, B; Boves, H; Holler, E

    1995-05-20

    beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days. The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63% homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences. Yields of beta-L-[14C]malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column. For most biochemical work, the product was partially purified with an overall 45% yield by chromatography on DEAE-Sephacel. The identity of the compound as beta-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart. The enzymatic synthesis, especially of radioactively labeled beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis. PMID:7573958

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

    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 P3212, 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 Å3 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

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

    The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. All five tRNALeu 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 tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS–tRNALeu 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 P21212, 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–tRNALeu, with a corresponding crystal volume per protein weight of 2.9 Å3 Da−1 and a solvent content of 57.3%

  18. Optimization of crude enzyme preparation methods for analysis of glutamine synthetase activity in phytoplankton and field samples

    WANG Yujue; WANG Dazhi; HONG Huasheng

    2009-01-01

    Glutamine synthetase (GS) is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation (HC, <10 000 g), ultracentrifugation (UC, 70 000 g), and ultrafiltration (UF) with 100 kμ, molecular weight cutoff, were compared using two diatom species (Asterionellopsis glacialis and Thalassiosira weissflogii), and two dinoflagellate species (Alexandrium catenella and Prorocentrum donghaiense) as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity (GSA) in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods (HC and UF) overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.

  19. Glutamine synthetase and Wnt-signaling%谷氨酰胺合成酶与Wnt信号转导通路

    彭春伟; 燕敏

    2009-01-01

    GS(glutamine synthetase)或GLUL(glutamate-ammonia ligase),即谷氨酰胺合成酶,为人体内重要的功能酶,催化谷氨酸与氨生成谷氨酰胺.在体内氮的代谢中,尤其在维持氨离子和谷氨酰胺的稳定中发挥着重要的作用.GS表达和活性的异常常会导致人体很多疾病的发生.近年来研究发现GS表达和活性的异常与Wnt信号通路的异常密切相关.%GS is also called glutamine synthetase or glutamate-ammonia ligase, which is an important enzyme in human body. It can catalyze glutamine from glutamate and ammonia. Besides, it plays an important role in nitrogen metabolism, particularly in homeostasis of blood levels of ammonium ions and glutamine. The aberrant expression and activity will cause many human diseases. Recent studies show that the abnormality of its expression and activity has a close relationship with the aberrance of Wnt-signaling.

  20. Crystallogenesis in tRNA aminoacylation systems: how packing accounts for crystallization drawbacks with yeast aspartyl-tRNA synthetase

    Sauter, C.; Lorber, B.; Théobald-Dietrich, A.; Giegé, R.

    2001-11-01

    Two active forms of homodimeric aspartyl-tRNA synthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P4 12 12) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 Å resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 Å 2, about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 Å 2, brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 Å 2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 Å 2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.

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

    Liron Klipcan

    2010-01-01

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

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

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

    2014-04-16

    The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions. PMID:24701966

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

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

    2012-09-01

    Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as Escherichia coli often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium Rhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report the apo (1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of RpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site of RpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity. PMID:22773649

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

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

    2016-01-01

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

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

    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.

  6. Bacterial Type I Glutamine Synthetase of the Rifamycin SV Producing Actinomycete, Amycolatopsis mediterranei U32, is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

    Wen-Tao PENG; Jin WANG; Ting WU; Jian-Qiang HUANG; Jui-Shen CHIAO; Guo-Ping ZHAO

    2006-01-01

    The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA+ gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.

  7. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix

    The editing domain of threonyl-tRNA synthetase from the archaeon Aeropyrum pernix has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.66 Å. The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P41212 or P43212 space group and consist of one monomer per asymmetric unit

  8. NPS6, Encoding a Non-Ribosomal Peptide Synthetase Involved in Siderophore-Mediated Iron Metabolism, is a Conserved Virulence Determinant of Plant Pathogenic Ascomycetes

    NPS6, encoding a non-ribosomal peptide synthetase, is a virulence determinant in the corn pathogen Cochliobolus heterostrophus and is also involved in resistance to oxidative stress, generated by hydrogen peroxide. Deletion of NPS6 orthologs in the rice pathogen, Cochliobolus miyabeanus, the cereal...

  9. NITRIC OXIDE (NO, CITRULLINE - NO CYCLE ENZYMES, GLUTAMINE SYNTHETASE AND OXIDATIVE STRESS IN ANOXIA (HYPOBARIC HYPOXIA AND REPERFUSION IN RAT BRAIN

    M. Swamy, Mohd Jamsani Mat Salleh, K. N .S. Sirajudeen, Wan Roslina Wan Yusof, G. Chandran

    2010-01-01

    Full Text Available Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia and reperfusion (reoxygenation, the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite, thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex, cerebellum and brain stem of rats subjected to anoxia and reperfusion. The results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase. The increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia, making nitric oxide production more effective and contributing to its toxic effects. The decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia. The increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion. The increased arginase and sustained decrease of GS activity in reperfusion group likely to be protective.

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

    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.

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

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

    1994-01-01

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

  12. Construction of hybrid peptide synthetases for the production of alpha-l-aspartyl-l-phenylalanine, a precursor for the high-intensity sweetener aspartame.

    Duerfahrt, Thomas; Doekel, Sascha; Sonke, Theo; Quaedflieg, Peter J L M; Marahiel, Mohamed A

    2003-11-01

    Microorganisms produce a large number of pharmacologically and biotechnologically important peptides by using nonribosomal peptide synthetases (NRPSs). Due to their modular arrangement and their domain organization NRPSs are particularly suitable for engineering recombinant proteins for the production of novel peptides with interesting properties. In order to compare different strategies of domain assembling and module fusions we focused on the selective construction of a set of peptide synthetases that catalyze the formation of the dipeptide alpha-l-aspartyl-l-phenylalanine (Asp-Phe), the precursor of the high-intensity sweetener alpha-l-aspartyl-l-phenylalanine methyl ester (aspartame). The de novo design of six different Asp-Phe synthetases was achieved by fusion of Asp and Phe activating modules comprising adenylation, peptidyl carrier protein and condensation domains. Product release was ensured by a C-terminally fused thioesterase domains and quantified by HPLC/MS analysis. Significant differences of enzyme activity caused by the fusion strategies were observed. Two forms of the Asp-Phe dipeptide were detected, the expected alpha-Asp-Phe and the by-product beta-Asp-Phe. Dependent on the turnover rates ranging from 0.01-0.7 min-1, the amount of alpha-Asp-Phe was between 75 and 100% of overall product, indicating a direct correlation between the turnover numbers and the ratios of alpha-Asp-Phe to beta-Asp-Phe. Taken together these results provide useful guidelines for the rational construction of hybrid peptide synthetases. PMID:14622284

  13. Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex

    Zou, Zhiying; Tong, Fumin; Færgeman, Nils J.;

    2003-01-01

    In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions ...

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

    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: 1.806, year: 2014

  15. Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)

    Chakraburtty, Rekha; White, Janet; Takano, Eriko; Bibb, Mervyn

    1996-01-01

    An internal segment of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. relA lies downstream of a gene (apt) that apparently en

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

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

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

    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

  18. Reaction Mechanism and Structural Model of ADP-forming Acetyl-CoA Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus: EVIDENCE FOR A SECOND ACTIVE SITE HISTIDINE RESIDUE*S⃞

    Bräsen, Christopher; Schmidt, Marcel; Grötzinger, Joachim; Schönheit, Peter

    2008-01-01

    In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + Pi ⇆ acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain orga...

  19. Prokaryote phylogeny based on ribosomal proteins and aminoacyl tRNA synthetases by using the compositional distance approach

    WEI; Haibin; QI; Ji; HAO; Bailin

    2004-01-01

    In order to show that the newly developed K-string composition distance method,based on counting oligopeptide frequencies,for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data,we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species.The latter group has been known to yield inconsistent trees if used individually.Our trees are obtained without making any sequence alignment.Altogether 16 Archaea,105 Bacteria and 2 Eucarya are represented on the tree.Most of the lower branchings agree well with the latest,2003,Outline of the second edition of the Bergey's Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.

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

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown to be...... an isolated transcriptional unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may...... satisfy 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...

  1. A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Suzuki, Takehiro; Dohmae, Naoshi; Kakeya, Hideaki

    2015-12-01

    Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments. PMID:26467472

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

    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.

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

    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. PMID:25584894

  4. FERONIA receptor kinase interacts with S-adenosylmethionine synthetase and suppresses S-adenosylmethionine production and ethylene biosynthesis in Arabidopsis.

    Mao, Dandan; Yu, Feng; Li, Jian; Van de Poel, Bram; Tan, Dan; Li, Jianglin; Liu, Yanqionq; Li, Xiushang; Dong, Mengqiu; Chen, Liangbi; Li, Dongping; Luan, Sheng

    2015-12-01

    Environmental inputs such as stress can modulate plant cell metabolism, but the detailed mechanism remains unclear. We report here that FERONIA (FER), a plasma membrane receptor-like kinase, may negatively regulate the S-adenosylmethionine (SAM) synthesis by interacting with two S-adenosylmethionine synthases (SAM1 and SAM2). SAM participates in ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for protein and DNA methylation reactions. The Arabidopsis fer mutants contained a higher level of SAM and ethylene in plant tissues and displayed a dwarf phenotype. Such phenotype in the fer mutants was mimicked by over-expressing the S-adenosylmethionine synthetase in transgenic plants, whereas sam1/2 double mutant showed an opposite phenotype. We propose that FER receptor kinase, in response to environmental stress and plant hormones such as auxin and BR, interacts with SAM synthases and down-regulates ethylene biosynthesis. PMID:25988356

  5. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis.

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E; Nikolaev, Evgeni V; Magni, Giulio; Zhang, Hong; Osterman, Andrei L

    2009-03-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-A resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN --> NMN --> NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN --> NaAD --> NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  6. Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

    Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana; Cho, Chris; Soll, Dieter; Simonovic, Miljan (Yale); (UIC)

    2014-03-12

    Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular ?? with a threonine (Thr) anticodon, MST1 also recognizes an unusual ??, which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for ?? recognition, whereas the anticodon sequence is essential for aminoacylation of ??. The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate ??, reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix {alpha}11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

  7. Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module.

    Yan, Wei; Ye, Qing; Tan, Min; Chen, Xi; Eriani, Gilbert; Wang, En-Duo

    2015-05-01

    A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. PMID:25817995

  8. Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

    Seabra Ana R

    2010-08-01

    Full Text Available Abstract Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS, occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. Results This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2 in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. Conclusions This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate

  9. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

    Romans Fuertes, Patricia; Søndergaard, Teis Esben; Sandmann, Manuela Ilse Helga;

    2016-01-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens- mediated transformation (ATMT) approach to generate...... knock-out mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway...... biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium...

  10. Sequence characterization and computational analysis of the non-ribosomal peptide synthetases controlling biosynthesis of lipopeptides, fengycins and bacillomycin D, from Bacillus amyloliquefaciens Q-426.

    Zhao, Pengchao; Quan, Chunshan; Jin, Liming; Wang, Lina; Guo, Xinjuan; Fan, Shengdi

    2013-12-01

    Lipopeptides secreted by bacteria attract interest because of their uses in biomedicine, biotechnology and food technology; however, harnessing their megasynthases (non-ribosomal peptide synthetases, NRPSs) has met with some difficulties in heterologous expression and crystallization. Here, we used similarity and phylogenetic analysis of NRPS sequences, including the fengycin and iturin family synthetases from Bacillus spp., and have developed a novel approach for delineating the length and boundaries of NRPS domains from Bacillus amyloliquefaciens strain Q-426. The sequences were further characterized (including specific residues and conserved motifs) that gave insight into the basis of the substrate specificity. Data from the prediction of the NRPS domains, obtained by the self-optimized prediction method with Alignment program, showed they are all structurally unstable, making it difficult to determine their crystal structures. PMID:24068498

  11. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

    Hove-Jensen, Bjarne

    1985-01-01

    deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells...... by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent...... and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion...

  12. NITRIC OXIDE (NO), CITRULLINE - NO CYCLE ENZYMES, GLUTAMINE SYNTHETASE AND OXIDATIVE STRESS IN ANOXIA (HYPOBARIC HYPOXIA) AND REPERFUSION IN RAT BRAIN

    M. Swamy, Mohd Jamsani Mat Salleh, K. N .S. Sirajudeen, Wan Roslina Wan Yusof, G. Chandran

    2010-01-01

    Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia) and reperfusion (reoxygenation), the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase...

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

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

    2014-01-01

    Coarse-grained simulations have emerged as invaluable tools for studying conformational changes in biomolecules. To evaluate the effectiveness of computationally inexpensive coarse-grained models in studying global and local dynamics of large protein systems like aminoacyl-tRNA synthetases, we have performed coarse-grained normal mode analysis, as well as principle component analysis on trajectories of all-atom and coarse-grained molecular dynamics simulations for three aminoacyl-tRNA synthet...

  14. Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

    Bayat, Vafa; Thiffault, Isabelle; Jaiswal, Manish; Tétreault, Martine; Donti, Taraka; Sasarman, Florin; Bernard, Geneviève; Demers-Lamarche, Julie; Dicaire, Marie-Josée; Mathieu, Jean; Vanasse, Michel; Bouchard, Jean-Pierre; Rioux, Marie-France; Lourenco, Charles M; Li, Zhihong

    2012-01-01

    An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photo...

  15. Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

    Ramos, F R; López-Nieto, M J; Martín, J F

    1985-01-01

    The tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicillium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2+ and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25 degrees C and 7.8, respectively. The reaction required O2 and was stimulated by increasing the diss...

  16. A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.

    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.

  17. Repercussion of mesophyll-specific overexpression of a soybean cytosolic glutamine synthetase gene in alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.)

    Seger, Mark; Ortega, Jose Luis; Bagga, Suman; Gopalan, Champa-Sengupta

    2009-01-01

    Glutamine synthetase (GS) plays a central role in plant nitrogen metabolism. Plant GS occurs as a number of isoenzymes present in either the cytosol (GS1) or chloroplast/plastid (GS2). There are several reports of improved performance in transgenic plants overexpressing GS1 transgenes driven by the constitutive CaMV35S promoter. Improvement has been attributed to the GS1 transgene product functioning to enhance re-assimilation of NH4+ released by photorespiration or protein degradation. In th...

  18. Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes

    Lorenzi, Adriana Sturion; Silva, Genivaldo Gueiros Z.; Lopes, Fabyano Alvares Cardoso; Chia, Mathias Ahii; Edwards, Robert A.

    2016-01-01

    Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes). PMID:27151783

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

    The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. [3H]Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly

  20. Changes in Activities of Key Enzymes for Starch Synthesis and Glutamine Synthetase in Grains of Progenies from a Rice Cross During Grain Filling

    LI Xiao-guang; LIU Hai-ying; JIN Zheng-xun; LIU Hong-liang; HUANG Xing; XU Mei-lan; ZHANG Feng-zhuan

    2010-01-01

    The progenies differed in amylose and protein contents in grains, which derived from a rice cross, Dongnong 423×Toukei 180, were used to study changes in the activities of ADP-glucose pyrophosphorylase (AGPP), soluble starch synthetase (SSS), starch branching enzyme (SBE) and glutamine synthetase (GS) in rice grains during grain filling. The activities of AGPP, SSS and SBE gradually increased and then declined as a single-peak curve with the process of grain filling in the progenies with high and low amylose contents in grains. The progenies with high amylose content peaked earlier in the AGPP, SSS and SBE activities and had higher AGPP, SSS and SBE activities at the early grain filling stage than those with low amylose content. The GS activity peaked earlier and was higher at the late stage of grain filling in the progenies with high protein content than in those with low protein content. It is suggested that the activities of key enzymes for starch synthesis and glutamine synthetase could be changed in oriented breeding for amylose and protein contents in grains.

  1. Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

    Antonella Albino

    2011-09-01

    Full Text Available Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from L-γ-glutamyl-L-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P212121, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.

  2. The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE

    Francisco, Brian San; Sutherland, Molly C.; Kranz, Robert G.

    2014-01-01

    SUMMARY Cytochrome c maturation (ccm) in many bacteria, archaea, and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches heme covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of heme to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to heme. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2, and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis. PMID:24397552

  3. Ammonium assimilation in rice based on the occurrence of 15N and inhibition of glutamine synthetase activity

    Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two-week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX) an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N-labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gin) and glutamic acid (Glu) before being converted to other amino acids through transamination. The formation of amino acids such as aspartic acid (Asp) and alanine (Ala) directly from free NH4 in roots also has been reported. Translocation of free NH4 to plant shoots, based on the concentration of free NH4 in xylem exudate, has been reported in tomato, although NH4 in shoots primarily originates from nitrate reduction in the shoot. Photorespiration also can contribute to the accumulation of NH4 in leaves. The GS/GOGAT cycle appears to be primarily responsible for the assimilation of exogenously supplied NH4 and NH4 derived from nitrate reduction in leaves, as well as NH4 derived from photorespiration. Genetic evidence cited to support this conclusion includes the lethal effect of photorespiratory conditions on plant mutants deficient in chloroplast-localized GS and GOGAT activities, and the rapid accumulation of free NH4 in GS-deficient mutants under photorespiratory conditions. The present study was initiated to quantify the in vivo amino acid synthesis in rice

  4. Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases.

    Narita, Tomomi; Naganuma, Tatsuro; Sase, Yurie; Kihara, Akio

    2016-01-01

    Transport of dietary lipids into small-intestinal epithelial cells is pathologically and nutritionally important. However, lipid uptake remains an almost unexplored research area. Although we know that long-chain bases (LCBs), constituents of sphingolipids, can enter into cells efficiently, the molecular mechanism of LCB uptake is completely unclear. Here, we found that the yeast acyl-CoA synthetases (ACSs) Faa1 and Faa4 are redundantly involved in LCB uptake. In addition to fatty acid-activating activity, transporter activity toward long-chain fatty acids (LCFAs) has been suggested for ACSs. Both LCB and LCFA transports were largely impaired in faa1Δ faa4Δ cells. Furthermore, LCB and LCFA uptakes were mutually competitive. However, the energy dependency was different for their transports. Sodium azide/2-deoxy-D-glucose treatment inhibited import of LCFA but not that of LCB. Furthermore, the ATP-AMP motif mutation FAA1 S271A largely impaired the metabolic activity and LCFA uptake, while leaving LCB import unaffected. These results indicate that only LCFA transport requires ATP. Since ACSs do not metabolize LCBs as substrates, Faa1 and Faa4 are likely directly involved in LCB transport. Furthermore, we revealed that ACSs are also involved in LCB transport in mammalian cells. Thus, our findings provide strong support for the hypothesis that ACSs directly transport LCFAs. PMID:27136724

  5. STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

    de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

    2016-07-15

    Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. PMID:27190175

  6. Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes▿

    van der Knaap, Jan A.; Kozhevnikova, Elena; Langenberg, Karin; Moshkin, Yuri M.; Verrijzer, C. Peter

    2010-01-01

    Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors. PMID:19995917

  7. Tumor-suppressive functions of long-chain acyl-CoA synthetase 4 in gastric cancer.

    Ye, Xiaojuan; Zhang, Yi; Wang, Xiao; Li, Yandong; Gao, Yong

    2016-04-01

    Long chain acyl CoA synthetase 4 (ACSL4) is a key enzyme in fatty acid metabolism with marked preference for arachidonic acid (AA). Recent reports have implicated its crucial roles in tumorigenesis. However in gastric cancer (GC), the expression and function of ACSL4 remain unclear. In the present study, we identified ACSL4 as a potential tumor suppressor in GC. The ACSL4 expression in GC samples was evaluated by real-time PCR and immunohistochemistry. The results indicated that the mRNA and protein levels of ACSL4 were frequently downregulated in cancer tissues compared with the adjacent non-cancerous mucosa control tissues. Cell-based functional assays exhibited that ectopic expression of ACSL4 inhibits cell growth, colony formation and cell migration, whereas ACSL4 knockdown enhanced these effects. In a nude mice model, ACSL4 knockdown also promoted subcutaneous xenografts' growth in vivo. Moreover, western blot analysis revealed that ACSL4 expression had a significant effect on FAK and P21 protein level. These findings suggest that ACSL4 plays a tumor-suppressive role and could be a potential therapeutic target in GC. PMID:26949059

  8. Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids

    Tyler J. Ford

    2015-06-01

    Full Text Available FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6–12 carbons are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

  9. Study of the Binding Energies between Unnatural Amino Acids and Engineered Orthogonal Tyrosyl-tRNA Synthetases

    Ren, Wei; Truong, Tan M.; Ai, Hui-Wang

    2015-07-01

    We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs.

  10. CDP-diacylglycerol synthetase coordinates cell growth and fat storage through phosphatidylinositol metabolism and the insulin pathway.

    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.

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

    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. PMID:25858017

  12. Studies on Glutamine Synthetase Activity in Sugar Beet(Beta vulgaris L.) under Different Levels of Nitrogen

    YanGuiping; YanHui; 等

    1995-01-01

    It was shown from the experiment that glutamine synthetase activity (GSA) in both leaf blades and roots under different nitrogen levels rose rapidly to reach its peak from seeding stage to foliage rapid growth stage and declined to its lowest level at the latter stage of root rapid growth,and then increased slightly,GSA in leaf blades had positive correlation with nitrogen level during the whole period of growth,GSA in roots showed the same tendency as it in leaf blades at the early middle stage of growth,but at the latter stage of growth,no positive correlation was established.GSA in leaf blades was the strongest compared with crowns,petioles and roots,and could represent the highest enzyme activity of the whole pant,GSA had quadratic curvilinear correlation with root yield and sugar production.GSA in leaf blades had significant positive correlation with α-NH2-N at the foliage rapid growth stage.

  13. Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase.

    Kwong, L K; Moore, V G; Kaiser, I I

    1977-09-25

    Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme. PMID:330528

  14. New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

    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.

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

    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.

  16. Analysis of the Resistance Mechanism of a Benzoxaborole Inhibitor Reveals Insight into the Leucyl-tRNA Synthetase Editing Mechanism.

    Zhao, Hanchao; Palencia, Andres; Seiradake, Elena; Ghaemi, Zhaleh; Cusack, Stephen; Luthey-Schulten, Zaida; Martinis, Susan

    2015-10-16

    A new class of antimicrobial benzoxaborole compounds was identified as a potent inhibitor of leucyl-tRNA synthetase (LeuRS) and therefore of protein synthesis. In a novel mechanism, AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) blocks fungal cytoplasmic LeuRS by covalently trapping tRNA(Leu) in the editing site of the enzyme's CP1 domain. However, some resistant mutation sites are located outside of the CP1 hydrolytic editing active site. Thus, their mode of action that undermines drug inhibition was not understood. A combination of X-ray crystallography, molecular dynamics, metadynamics, biochemical experiments, and mutational analysis of a distal benzoxaborole-resistant mutant uncovered a eukaryote-specific tyrosine "switch" that is critical to tRNA-dependent post-transfer editing. The tyrosine "switch" has three states that shift between interactions with a lysine and the 3'-hydroxyl of the tRNA terminus, to inhibit or promote post-transfer editing. The oxaborole's mechanism of action capitalizes upon one of these editing active site states. This tunable editing mechanism in eukaryotic and archaeal LeuRSs is proposed to facilitate precise quality control of aminoacylation fidelity. These mechanistic distinctions could also be capitalized upon for development of the benzoxaboroles as a broad spectrum antibacterial. PMID:26172575

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

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

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. PMID:26079224

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

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi. PMID:20053634

  19. Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis.

    Watford, Malcolm; Wu, Guoyao

    2005-04-01

    High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07+/-0.01 U/g) when compared to leg muscle (0.50+/-0.04 U/g). Free glutamine levels were 1.38+/-0.09 and 9.69+/-0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82+/-0.35 nmol/mg wet weight) when compared to leg muscle (16.2+/-1.0 nmol/mg wet weight) and much lower (1.80+/-0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content. PMID:15763516

  20. Purification and properties of the fatty acids synthetase complex from Neurospora crassa, and the nature of the fas-mutation.

    Elovson, J

    1975-01-01

    A procedure is described for the purification of the fatty acid synthetase complex (FAS) from Neurospora crassa. The enzyme complex has a molecular weight of 2.3 times 10(6), contains 6 mol of 4'-phosphopantetheine per mol, and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a single band, or a closely spaced doublet, which comigrates with standard myosin (molecular weight, 2 times 10(5)). Since the slightly retarded component in the doublet accounts for all protein-bound 4'-phosphopantetheine, the complex appears to be made up of 11 to 12 equally sized subunits, 6 of which carry the acyl carrier protein function. In this unusual arrangement, notably the lack of the low-molecular-weight acyl carrier protein component seen in other FAS systems, as well as in its enzymatic properties, the Neurospora FAS complex is quite similar to the yeast enzyme. The FAS complex of a saturated fatty acid-requiring mutant, previously disignated cel-, contains less than 2% of the 4'-phosphopantetheine prosthetic groups found in the wild-type complex. The leaky phenotype of this mutant, here designated fas-, is accounted for by a residual fatty acid synthesizing activity in its FAS complex, which is several-fold higher than expected from its residual content of 4'-phosphopanthetheine. Images PMID:126228

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

    Knight, T.J. [Univ. of Southern Maine, Portland, ME (United States); Temple, S.; Sengupta-Gopalan, C. [New Mexico State Univ., Las Curces, NM (United States)] [and others

    1996-05-15

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

  2. The T1405N carbamoyl phosphate synthetase polymorphism does not affect plasma arginine concentrations in preterm infants.

    Rob M J Moonen

    Full Text Available BACKGROUND: A C-to-A nucleotide transversion (T1405N in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1 has been associated with changes in plasma concentrations of L-arginine in term and near term infants but not in adults. In preterm infants homozygosity for the CPS1 Thr1405 variant (CC genotype was associated with an increased risk of having necrotizing enterocolitis (NEC. Plasma L-arginine concentrations are decreased in preterm infants with NEC. AIM: To examine the putative association between the CPS1 T1405N polymorphism and plasma arginine concentrations in preterm infants. METHODS: Prospective multicenter cohort study. Plasma and DNA samples were collected from 128 preterm infants (<30 weeks between 6 and 12 hours after birth. Plasma amino acid and CPS1 T1405N polymorphism analysis were performed. RESULTS: Distribution of genotypes did not differ between the preterm (CC:CA:AA = 55.5%:33.6%:10.9%, n = 128 and term infants (CC:CA:AA = 54.2%:35.4%:10.4%, n = 96. There was no association between the CPS1 genotype and plasma L-arginine or L-citrulline concentration, or the ornithine to citrulline ratio, which varies inversely with CPS1 activity. Also the levels of asymmetric dimethylarginine, and symmetric dimethylarginine were not significantly different among the three genotypes. CONCLUSIONS: The present study in preterm infants did not confirm the earlier reported association between CPS1 genotype and L-arginine levels in term infants.

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

    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.

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

    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.

  5. The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

    Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf; Neuhof, Torsten; Baker, Scott E.; Druzhinina, Irina S.; Kubicek, Christian P.; Brückner, Hans; von Dohren, Hans

    2012-03-01

    The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.

  6. TXNIP Suppresses Expression of Glutamine Synthetase by Inducing Oxidative Stress in Retinal Muller Glia Under Diabetic Conditions.

    Zhou, Jia; Shen, Xi; Lu, Qiong; Zhang, Min

    2016-01-01

    BACKGROUND Diabetic retinopathy (DR) is a progressive neurodegenerative disease with early-stage symptoms such as dysfunction of Muller cells, which leads to ganglion cell death. Its pathogenesis is probably associated with oxidative stress and a recently discovered protein, thioredoxin-interacting protein (TXNIP). MATERIAL AND METHODS To explore the role of TXNIP in DR, we cultured Muller cells under diabetic conditions, and then used immunohistochemistry, Western blot, and RT-PCR to detect the expression level of TXNIP under diabetic conditions. We demonstrated the expression level of glutamine synthetase (GS) when TXNIP was inhibited. To explore the potential pathway of TXNIP-induced cell damage in DR, we confirmed the role of IL-1β under diabetic conditions. RESULTS Diabetes induces TXNIP expressions at mRNA levels, but shows the opposite effect on GS. IL-1β plays an important role in this pathway. Azaserine effectively increased the expression of GS via attenuating the expression of TXNIP. CONCLUSIONS This study demonstrates the role of TXNIP and its mechanism in DR, provides a possible treatment for DR, and lays a new theoretical foundation for the clinical treatment of DR and other diabetic microvascular changes. PMID:27131835

  7. BIOTRANSFORMATION USING RECOMBINANT CMP SIALIC ACID SYNTHETASE AND α-2, 6-SIALYLTRAN SFERASE: ENZYMATIC SYNTHESIS OF SIALOSIDES

    Ulrike Hubl

    2012-01-01

    Full Text Available In this research, we successfully expressed recombinant CMP-sialic Acid Synthetase (CSS from Neisseria meningitides and 2,6-Sialyltransferase (SAT from Photobacterium damsela in E. coli BL21(DE3 fermented at a scale of up to 8 litres using individual plasmids pIRL-1 and pIRL-4b, respectively. After cell lysis with BugBuster, enzyme levels of 2U and 22U per litre were produced for CSS and SAT, respectively. The enzyme solutions were either used directly as crude preparations or further purified by affinity chromatography. Characterization of the CSS and SAT confirmed that both enzymes had comparable properties to those described in the literature. The production of cytidine 5’-monophosphate N-acetylneuraminic acid (CMP-NeuAc and CMP-9-azido-NeuAc using crude CSS was successful with >90% conversion at scales from 100 mg to 5 g. Activated sugar purification by ethanol precipitation was optimized. Finally, the CSS and SAT enzymes were applied to a large-scale synthesis of a sialylated lactosamine glycoside via a two-step biotransformation. The initial step employed crude CSS to convert Cytidine Triphosphate (CTP and 9-azido-NeuAc to CMP-9-azido-NeuAc at a conversion efficiency of 98%. This reaction mixture, after ultrafiltration to remove β-galactosidase activity co-expressed by E. coli BL21, was used as the donor substrate for the second step involving SAT. The sialoside 9-azido-sialyl-α-2,6’-lactosamine glycoside was produced with 86% conversion of the starting glycoside. Purification of the product was achieved by chromatography on Diaion HP-20 (a hydrophobic styrenic resin.

  8. Downregulation of glutamine synthetase via GLAST suppression induces retinal axonal swelling in a rat ex vivo hydrostatic pressure model.

    Ishikawa, Makoto; Yoshitomi, Takeshi; Zorumski, Charles F; Izumi, Yukitoshi

    2011-08-01

    PURPOSE. High levels of glutamate can be toxic to retinal GCs. Thus, effective buffering of extracellular glutamate is important in preserving retinal structure and function. GLAST, a major glutamate transporter in the retina, and glutamine synthetase (GS) regulate extracellular glutamate accumulation and prevent excitotoxicity. This study was an examination of changes in function and expression of GLAST and GS in ex vivo rat retinas exposed to acute increases in ambient pressure. METHODS. Ex vivo rat retinas were exposed to elevated hydrostatic pressure for 24 hours. The expression of GLAST and GS were examined using immunochemistry and real-time PCR analysis. Also examined were the effects of (2S,3S)-3-[3-[4-(trifluoromethyl) benzoylamino] benzyloxy] aspartate (TFB-TBOA), an inhibitor of glutamate transporters, and l-methionine-S-sulfoximine (MSO), an inhibitor of GS. RESULTS. In this acute model, Western blot and real-time RT-PCR analyses revealed that substantially (75 mm Hg), but not moderately (35 mm Hg), elevated pressure depressed GLAST expression, diminished GS activity, and induced axonal swelling between the GC layer and the inner limiting membrane. However, at the moderately elevated pressure (35 mm Hg), administration of either TFB-TBOA or MSO also induced axonal swelling and excitotoxic neuronal damage. MSO did not depress GLAST expression but TFB-TBOA significantly suppressed GS, suggesting that downregulation of GS during pressure loading may result from impaired GLAST expression. CONCLUSIONS. The retina is at risk during acute intraocular pressure elevation due to downregulation of GS activity resulting from depressed GLAST expression. PMID:21775659

  9. Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

    Uheda, Eiji; Maejima, Kazuhiro

    2009-10-15

    In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly. PMID:19464754

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

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

    2016-01-01

    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. PMID:27092515

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

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

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

    2016-04-01

    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. PMID:27092515

  13. Lethality of glnD null mutations in Azotobacter vinelandii is suppressible by prevention of glutamine synthetase adenylylation.

    Colnaghi, R; Rudnick, P; He, L; Green, A; Yan, D; Larson, E; Kennedy, C

    2001-05-01

    GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridylylates two related proteins, PII and GlnK. The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC. Results presented here demonstrate that glnD is an essential gene in Azotobacter vinelandii. Null glnD mutations were introduced into the A. vinelandii genome, but none could be stably maintained unless a second mutation was present that resulted in unregulated activity of GS. One mutation, gln-71, occurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein. The second, created by design, was glnAY407F (MV75), altering the adenylylation site of GS. The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia coli glnE gene into MV72, a glnD(+) derivative of MV71, restored the regulation of GS activity. GlnK-UMP is therefore apparently required for GS to be sufficiently deadenylylated in A. vinelandii for growth to occur. The DeltaglnD GS(c) isolates were Nif(-), which could be corrected by introducing a nifL mutation, confirming a role for GlnD in mediating nif gene regulation via some aspect of the NifL/NifA interaction. MV71 was unexpectedly NtrC(+), suggesting that A. vinelandii NtrC activity might be regulated differently than in enteric organisms. PMID:11320130

  14. Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

    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.

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

    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.

  16. Stage-dependent expression and up-regulation of trypanothione synthetase in amphotericin B resistant Leishmania donovani.

    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. AAE13 encodes a dual-localized malonyl-CoA synthetase that is crucial for mitochondrial fatty acid biosynthesis.

    Guan, Xin; Nikolau, Basil J

    2016-03-01

    Malonyl-CoA is a key intermediate in a number of metabolic processes associated with its role as a substrate in acylation and condensation reactions. These types of reactions occur in plastids, the cytosol and mitochondria, and although carboxylation of acetyl-CoA is the known mechanism for generating the distinct plastidial and cytosolic pools, the metabolic origin of the mitochondrial malonyl-CoA pool is still unclear. In this study we demonstrate that malonyl-CoA synthetase encoded by the Arabidopsis AAE13 (AT3G16170) gene is localized in both the cytosol and the mitochondria. These isoforms are translated from two types of transcripts, one that contains and one that does not contain a mitochondrial-targeting pre-sequence. Whereas the cytosolic AAE13 protein is not essential, due to the presence of a redundant malonyl-CoA generating system provided by a cytosolic acetyl-CoA carboxylase, the mitochondrial AAE13 protein is essential for plant growth. Phenotypes of the aae13-1 mutant are transgenically reversed only if the mitochondrial pre-sequence is present in the ectopically expressed AAE13 proteins. The aae13-1 mutant exhibits typical metabolic phenotypes associated with a deficiency in the mitochondrial fatty acid synthase system, namely depleted lipoylation of the H subunit of the photorespiratory enzyme glycine decarboxylase, increased accumulation of glycine and glycolate and reduced levels of sucrose. Most of these metabolic alterations, and associated morphological changes, are reversed when the aae13-1 mutant is grown in a non-photorespiratory condition (i.e. a 1% CO2 atmosphere), demonstrating that they are a consequence of the deficiency in photorespiration due to the inability to generate lipoic acid from mitochondrially synthesized fatty acids. PMID:26836315

  18. Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

    Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-07-01

    Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production. PMID:26996628

  19. Elucidating the Pseudomonas aeruginosa fatty acid degradation pathway: identification of additional fatty acyl-CoA synthetase homologues.

    Jan Zarzycki-Siek

    Full Text Available The fatty acid (FA degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300, which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924 were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617 contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.

  20. Elucidating the Pseudomonas aeruginosa fatty acid degradation pathway: identification of additional fatty acyl-CoA synthetase homologues.

    Zarzycki-Siek, Jan; Norris, Michael H; Kang, Yun; Sun, Zhenxin; Bluhm, Andrew P; McMillan, Ian A; Hoang, Tung T

    2013-01-01

    The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h. PMID:23737986

  1. Effects of aerosolized ketamine on the level of nitric oxide and nitric oxide synthetase in the lung tissue of rat with asthma

    2006-01-01

    Objective: To explore the effects of aerosolized ketamine on the level of nitric oxide and nitric oxide synthetase in the lung tissue in rat asthma model. Methods: Forty SD rats were randomly assigned to five groups: control group (group N), asthma model group (group A), two pretreated groups of different concentrations of ketamine (group K1, K2)and dexamethasone group(group D) with eight rats in each group. The rats in group A were sensitized by injection of ovalbumin (OA) together with aluminum hydroxide and bordetella pertussis as adjuvants. Two weeks after the sensitization, aerosolized OA was used to cause asthma. The rats in group K1 and K2 were sensitized with OA as group A , and then exposed to aerosol of ketamine , with the concentration of 25 g/L and 50 g/L respectively. Before using aerosolized OA, the rats in group D were exposed to aerosol of 0.01% dexamethasone . The level of NO2-/NO3- in lung tissues, inducible nitric oxide synthetase(iNOS) and constitute nitric oxide synthetase(cNOS) was measured in all groups. Results: The level of NO2-/NO3- and the activity of iNOS in lung tissues in group A were signiticantly higher than those in the other groups. The iNOS activity and the level of NO2-/NO3- in lung tissues were highly positively correlated. Conclusion: NO can induce airway hyperreactivity that may worsen asthma. Aerosolized ketamine can decrease the iNOS expression and reduce the level of NO in the lung tissue in rat asthma model.

  2. Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic.

    Trauger, J W; Walsh, C T

    2000-03-28

    The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins. PMID:10716695

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

    Pinyi Lu

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

  4. Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae

    Glutamyl-tRNA synthetase (GluRS) is an essential enzyme for protein translation and has been considered as a target for drug development. In this paper, the cloning, expression and purification of the GluRS protein encoded by the gltX gene (Xoo1504) and its preliminarily crystallographic analysis are reported. The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNAGlu. It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 Å resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 Å, β = 96.3°. The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (VM) range of 2.70–2.02 Å3 Da−1 and a solvent-content range of 54.5–39.3%

  5. GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light, plant hormones and auxinic herbicides.

    Ostrowski, Maciej; Jakubowska, Anna

    2013-03-01

    The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species. PMID:23332498

  6. Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka

    Schousboe, Mette L; Rajakaruna, Rupika S; Salanti, Ali;

    2007-01-01

    BACKGROUND: Single nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights into the...... resistance across Sri Lanka. SUBJECTS AND METHODS: P. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene...... and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure....

  7. Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

    Yang, Zhengrong W.; Tendian, Susan W.; Carson, W. Michael; Brouillette, Wayne J.; DeLucas, Lawrence J.; Brouillette, Christie G

    2004-01-01

    Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD+ synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme’s catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. ...

  8. Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli.

    Krause, M.; Marahiel, M A; von Döhren, H; Kleinkauf, H

    1985-01-01

    Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-...

  9. 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

    Nielsen, B; Hokland, M; Justesen, J;

    1989-01-01

    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...... treatment these effects were gradually abolished, indicating an increasing effect of IFN-alpha in vivo with time. These results shows that the different PBMNC subpopulations and important immunological functions normalize with treatment. This normalization is, however, not seen until at least after 1 year...

  10. 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?

    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

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

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.; Larsen, K.; Berthet-Colominas, C.; Leberman, R.; Beijer, B.; Sproat, B.; Als-Nielsen, J.; Grübel, G.; Legrand, J.-F.; Lehmann, M.; Cusack, S.

    1994-01-01

    crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a......Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the...

  12. Transcription Activity of Individual rrn Operons in Bacillus subtilis Mutants Deficient in (p)ppGpp Synthetase Genes, relA, yjbM, and ywaC▿ †

    Natori, Yousuke; Tagami, Kazumi; Murakami, Kana; Yoshida, Sawako; Tanigawa, Osamu; Moh, Yoonsuh; MASUDA, KENTA; Wada, Tetsuya; Suzuki, Shota; Nanamiya, Hideaki; Tozawa, Yuzuru; Kawamura, Fujio

    2009-01-01

    In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In or...

  13. Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNASec in complex with seryl-tRNA synthetase

    Bacterial selenocysteine tRNA was crystallized as the heterologous complex with archaeal seryl-tRNA synthetase. X-ray diffraction was improved by introducing point mutations and heavy-atom labeling, and a 3.2 Å diffraction data set for phase determination was obtained from a platinum-labeled crystal. Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNASec) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNASec and its specific interaction with SerRS, the bacterial tRNASec from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNASec in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K2Pt(CN)4-soaked crystal

  14. A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity.

    Tan, Min; Yan, Wei; Liu, Ru-Juan; Wang, Meng; Chen, Xin; Zhou, Xiao-Long; Wang, En-Duo

    2012-04-15

    aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism. PMID:22292813

  15. Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of pyrrolysyl-tRNA synthetase from the methanogenic archaeon Methanosarcina mazei

    Pyrrolysyl-tRNA synthetase (PylRS) from M. mazei has been overexpressed in an N-terminally truncated form PylRS(c270) in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. Pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei was overexpressed in an N-terminally truncated form PylRS(c270) in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The native PylRS(c270) crystals in complex with an ATP analogue belonged to space group P64, with unit-cell parameters a = b = 104.88, c = 70.43 Å, α = β = 90, γ = 120°, and diffracted to 1.9 Å resolution. The asymmetric unit contains one molecule of PylRS(c270). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method

  16. Small-angle X-ray-scattering investigation and structural-model study of the fatty-acid synthetase from pig liver

    The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetase from this and other sources. When comparing small-angle X-ray-scattering curves calculated with these models and curves obtained from small-angle X-ray-scattering experiments carried out with the pig-liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and the functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an asymmetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other. (orig.)

  17. Biodistribution of 3,4-dihydro-5-[11c]methoxy-1(2h)-isoquinolinone, a potential PET tracer for poly(adp-ribose) synthetase

    Poly(adenosine diphosphate-ribose) synthetase (PARS) is a nuclear enzyme that is activated by deoxyribonucleic acid (DNA) strand breaks and participates in DNA repair. Excessive PARS activation, however, leads to cell death due to depletion of adenosine triphosphate (ATP). To evaluate whether it is possible to detect excessive activation of PARS with positron emission tomography (PET), we examined the pharmacokinetics of 3,4-dihydro-5-[11C]methoxy-1(2H)-isoquinolinone ([11C]MIQO), a potent poly(ADP-ribose) synthetase inhibitor, in the brain of rats and monkeys. Although the uptake of [11C]MIQO in the brain of normal rats was low, [11C]MIQO was rapidly incorporated into and then quickly washed out from the brain. The uptake of the radiotracer in the brain of normal monkeys was also low; however, [11C]MIQO gave a distribution image that differed from that of cerebral blood flow obtained by [15O]water-PET. No localization of [11C]MIQO in the brain of normal monkeys was observed. Low accumulation of some radioactivity was also observed in muscles surrounding the brain of monkeys, but did not seem to interfere with measurement of [11C]MIQO uptake in the brain with PET. Thus, detection of [11C]MIQO uptake with PET may be useful for detecting PARS activity in ischemic injury

  18. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

    SAICAR synthetase from P. horikoshii OT3 has been cloned, expressed, purified and crystallized. The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13 Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (VM) of 2.3 Å3 Da−1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides

  19. Inhibition of thromboxane A2 synthetase failed to limit myocardial infarct size in a rabbit ischemia-reperfusion model.

    Tsuchida, A; Miura, T; Ogawa, T; Iwamoto, T; Shimamoto, K; Iimura, O

    1991-02-01

    The role of thromboxane A2 (TXA2) in myocardial necrosis during coronary occlusion and reperfusion was investigated by using a new long-acting TXA2 synthetase inhibitor, DP1904. A rabbit coronary branch was occluded for 30 min and then reperfused for 72h. Infarct size and area at risk were determined histologically and by fluorescent particles, respectively, for 4 groups; a saline receiving control group (C group), a DP1904 treated group (DP group), a heparin treated group (H group), and a DP1904 plus heparin treated group (DP-H group). The H group and DP-H group were included to examine the influence of heparinization on the effect of DP1904. In the DP and DP-H groups, 10 mg/kg of DP1904 was injected i.v. 2h before coronary occlusion, as well as 24 and 48h after reperfusion. This dose of DP1904 (10 mg/kg i.v.) was able to inhibit serum thromboxane B2 formation ex vivo to 1.1% of the control level 2h after its administration, and to 39.5% at 24h, in the rabbit (n = 5). The H and DP-H groups received 1000 units of heparin i.v. 3 min prior to coronary occlusion. The size of the area at risk, heart rate, blood pressure, and rate-pressure products were comparable between the 4 groups. Mortality was not significantly different in any group. Myocardial infarct size as the percentage of area at risk was 43.6 +/- 3.9% in C group (n = 10), 41.1 +/- 4.4% in DP group (n = 9), 47.8 +/- 3.0% in H group (n = 13), and 44.7 +/- 4.0% in DP-H group (n = 10), which were not significantly different. These findings suggest that TXA2 does not contribute directly to myocardial necrosis during coronary occlusion and reperfusion in the rabbit. PMID:2020088

  20. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

    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

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

    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

  2. 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

    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.

  3. 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

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

    2001-01-01

    Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p are esse...

  4. 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

    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 a...

  5. Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNA(Sec) in complex with seryl-tRNA synthetase.

    Itoh, Yuzuru; Sekine, Shun Ichi; Yokoyama, Shigeyuki

    2012-06-01

    Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal. PMID:22684069

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

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

    2015-01-01

    protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, Acsl1(T-/-) hearts contained 3 times more mitochondria with abnormal structure and displayed a 35-43% lower respiratory function. To study the effects of mTORC1 activation on mitochondrial structure......, which was likely caused by the 33-51% lower ATP synthase activity present in both vehicle- and rapamycin-treated Acsl1(T-/-) hearts. The turnover of microtubule associated protein light chain 3b in Acsl1(T-/-) hearts was 88% lower than controls, indicating a diminished rate of autophagy. Rapamycin...... treatment increased autophagy to a rate that was 3.1-fold higher than in controls, allowing the formation of autophagolysosomes and the clearance of damaged mitochondria. Thus, long-chain acyl-CoA synthetase isoform 1 (ACSL1) deficiency in the heart activated mTORC1, thereby inhibiting autophagy and...

  7. Cytosolic glutamine synthetase is important for photosynthetic efficiency and water use efficiency in potato as revealed by high-throughput sequencing QTL analysis

    Kaminski, Kacper Piotr; Sørensen, Kirsten Kørup; Andersen, Mathias Neumann;

    2015-01-01

    isoforms of cytosolic glutamine synthase were located in the QTL at chromosome 1 suggesting a major contribution of this enzyme to photosynthetic efficiency and thus WUE in potato. Indeed, Glutamine synthetase enzyme activity of leaf extracts was measured and found to be correlated with contrasting WUE......Potato (Solanum tuberosum L.) closes its stomata at relatively low soil water deficits frequently encountered in normal field conditions resulting in unnecessary annual yield losses and extensive use of artificial irrigation. Therefore, unraveling the genetics underpinning variation in water use...... efficiency (WUE) of potato is important, but has been limited by technical difficulties in assessing the trait on individual plants and thus is poorly understood. In this study, a mapping population of potatoes has been robustly phenotyped, and considerable variation in WUE under well-watered conditions was...

  8. [Dependence of creatine kinase and glycogen synthetase activities of skeletal muscles on state of adenine nucleotide phosphorylation and cAMP metabolism].

    Iakovlev, N N; Chagovets, N R; Maksimova, L V

    1980-01-01

    Changes in the contents of adenine nucleotides, creatine phosphate, inorganic phosphate, creatine, glucose-6-phosphate and glycogen and the activity of adenylate cyclase, creatine kinase, glycogen phosphorylase 31:51-AMP-phosphodiesterase and glycogen synthetase in muscles and of blood catecholamines were studied in adult rats before loading, immediately after the cessation of the muscular activity, and at rest. Adenine nucleotides are established to play a regulatory role in catabolic and anabolic processes nucleotides are established to play a regulatory role in catabolic and anabolic processes related to the muscular activity. It is established that compensation and supercompensation of the working losses of muscular creatine phosphate and glycogen are due to activation of anabolic processes under conditions of higher phosphorylation of the adenylic system. PMID:6247797

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

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

    2015-05-01

    Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities. PMID:25913036

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

    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 a...... significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by...

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

    Holmbeck, Marissa A; Donner, Julia R; Villa-Cuesta, Eugenia; Rand, David M

    2015-08-01

    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 simw(501) mtDNA-encoded transfer RNA (tRNA) for tyrosine (tRNA(Tyr)) 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 simw(501) 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 tRNA(Tyr) 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. PMID:26035388

  12. Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange

    The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O, 16O]Pi does not readily participate

  13. Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

    Wagner Martin

    2006-03-01

    Full Text Available Abstract Background Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS, but its exact genetic organization and biochemical synthesis is unknown. Results The complete sequence of the cereulide synthetase (ces gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. Conclusion The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces

  14. Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.

    Cho Yeow Koh

    2014-04-01

    Full Text Available Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general 'ATP-engaging' binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.

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

    Douglas S. Kernodle

    2013-01-01

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

  16. 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

    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)

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

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

  18. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from ∼ 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH

  19. Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence.

    Orsel, Mathilde; Moison, Michaël; Clouet, Vanessa; Thomas, Justine; Leprince, Françoise; Canoy, Anne-Sophie; Just, Jérémy; Chalhoub, Boulos; Masclaux-Daubresse, Céline

    2014-07-01

    A total of 16 BnaGLN1 genes coding for cytosolic glutamine synthetase isoforms (EC 6.3.1.2.) were found in the Brassica napus genome. The total number of BnaGLN1 genes, their phylogenetic relationships, and genetic locations are in agreement with the evolutionary history of Brassica species. Two BnaGLN1.1, two BnaGLN1.2, six BnaGLN1.3, four BnaGLN1.4, and two BnaGLN1.5 genes were found and named according to the standardized nomenclature for the Brassica genus. Gene expression showed conserved responses to nitrogen availability and leaf senescence among the Brassiceae tribe. The BnaGLN1.1 and BnaGLN1.4 families are overexpressed during leaf senescence and in response to nitrogen limitation. The BnaGLN1.2 family is up-regulated under high nitrogen regimes. The members of the BnaGLN1.3 family are not affected by nitrogen availability and are more expressed in stems than in leaves. Expression of the two BnaGLN1.5 genes is almost undetectable in vegetative tissues. Regulations arising from plant interactions with their environment (such as nitrogen resources), final architecture, and therefore sink-source relations in planta, seem to be globally conserved between Arabidopsis and B. napus. Similarities of the coding sequence (CDS) and protein sequences, expression profiles, response to nitrogen availability, and ageing suggest that the roles of the different GLN1 families have been conserved among the Brassiceae tribe. These findings are encouraging the transfer of knowledge from the Arabidopsis model plant to the B. napus crop plant. They are of special interest when considering the role of glutamine synthetase in crop yield and grain quality in maize and wheat. PMID:24567494

  20. The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a.

    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

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

    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.

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

    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. PMID:26443676

  3. Lengsin is a survivor of an ancient family of class I glutamine synthetases in eukaryotes that has undergone evolutionary re-engineering for a role in the vertebrate eye lens.

    Wyatt, Keith; White, Helen E.; Wang, Luchun; Bateman, Orval A.; Slingsby, Christine; Orlova, Elena V.; Wistow, Graeme

    2006-01-01

    Lengsin is a member of the glutamine synthetase (GS) superfamily expressed in the vertebrate eye lens. While the GS homology domains of lengsin are well conserved in all species examined, the N-terminal domain shows evidence of dynamic evolutionary changes. Compared with zebrafish and chicken, most mammals have an additional exon in the lengsin gene corresponding to part of the N-terminal domain, however in human this is a non-functional pseudoexon. Lengsin belongs to the hitherto purely prok...

  4. Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus.

    Alves-Bezerra, Michele; Klett, Eric L; De Paula, Iron F; Ramos, Isabela B; Coleman, Rosalind A; Gondim, Katia C

    2016-07-01

    Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6μM), but different Km for arachidonic acid (0.5 and 6μM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid β-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for β-oxidation in the fat body, and is also required for normal reproduction. PMID:27091636

  5. Molecular modeling of the reductase domain to elucidate the reaction mechanism of reduction of peptidyl thioester into its corresponding alcohol in non-ribosomal peptide synthetases

    Lee Gwang

    2010-01-01

    Full Text Available Abstract Background Nonribosomal peptide synthetases (NRPSs are multienzymatic, multidomain megasynthases involved in the biosynthesis of pharmaceutically important nonribosomal peptides. The peptaibol synthetase from Trichoderma virens (TPS is an important member of the NRPS family that exhibits antifungal properties. The majority of the NRPSs terminate peptide synthesis with the thioesterase (TE domain, which either hydrolyzes the thioester linkage, releasing the free peptic acid, or catalyzes the intramolecular macrocyclization to produce a macrolactone product. TPS is an important NRPS that does not encompass a TE domain, but rather a reductase domain (R domain to release the mature peptide product reductively with the aid of a NADPH cofactor. However, the catalytic mechanism of the reductase domain has not yet been elucidated. Results We present here a three-dimensional (3D model of the reductase domain based on the crystal structure of vestitone reductase (VR. VR belongs to the short-chain dehydrogenase/reductase (SDR superfamily and is responsible for the nicotinamide dinucleotide phosphate (NADPH-dependent reduction of the substrate into its corresponding secondary alcohol product. The binding sites of the probable linear substrates, alamethicin, trichotoxin, antiamoebin I, chrysopermin C and gramicidin, were identified within the modeled R domain using multiple docking approaches. The docking results of the ligand in the active site of the R domain showed that reductase side chains have a high affinity towards ligand binding, while the thioester oxygen of each substrate forms a hydrogen bond with the OH group of Tyr176 and the thiol group of the substrate is closer to the Glu220. The modeling and docking studies revealed the reaction mechanism of reduction of thioester into a primary alcohol. Conclusion Peptaibol biosynthesis incorporates a single R domain, which appears to catalyze the four-electron reduction reaction of a peptidyl

  6. Mechanism of Adherence of Streptococcus mutans to Smooth Surfaces I. Roles of Insoluble Dextran-Levan Synthetase Enzymes and Cell Wall Polysaccharide Antigen in Plaque Formation

    Mukasa, Hidehiko; Slade, Hutton D.

    1973-01-01

    The mechanism of adherence of Streptococcus mutans to smooth glass surfaces has been studied. The results with both viable and heat-killed cells showed that the process required (i) the synthesis of a water-insoluble dextran-levan polymer by cell-bound enzymes and (ii) the participation of a binding site on the surface of the S. mutans cell. Synthesis of the polymer from sucrose in the presence of the cells was required for adherence, and indicates that an “active” form of the polymer was required. Polymer synthesized by cell-free S. mutans enzymes when added to S. mutans cells did not produce adherence. Purified antibody globulin, specific for the a-d site in the polysaccharide S. mutans group a antigen, completely inhibited adherence. Antibody to the second antigen present in the polysaccharide molecule, the a antigen, did not inhibit adherence. The evidence indicates that adherence did not require an antigenic binding site which might be common to all S. mutans strains. The orientation of the synthetase enzyme(s), antigenic binding site, and dextran-levan polymer on the cell surface is under study. Images PMID:4582634

  7. Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4).

    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. PMID:22672155

  8. 2'-5' oligoadenylate synthetase-like 1 (OASL1) deficiency suppresses central nervous system damage in a murine MOG-induced multiple sclerosis model.

    Choi, Bo Young; Sim, Chan Kyu; Cho, Yeon Sook; Sohn, Min; Kim, Young-Joon; Lee, Myeong Sup; Suh, Sang Won

    2016-08-15

    Type I Interferon (IFN-I) is critical for antiviral and antitumor defense. Additionally, IFN-I has been used for treating multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system (CNS). Recently, we reported that 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulates IFN-I production upon viral infection and tumor challenge. Therefore, OASL1 deficient (Oasl1(-)(/)(-)) mice are resistant to viral infections and tumor challenge. In this study, we examined whether OASL1 plays a negative role in the development of autoimmune MS by using Oasl1(-)(/)(-) mice and a murine MS model, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Oasl1(-)(/)(-) mice showed enhanced resistance to EAE development compared to wild-type (WT) mice. Additionally, EAE-induced Oasl1(-)(/)(-) mice showed fewer infiltrated immune cells such as T cells and macrophages in the CNS and less CNS inflammation, compared to WT mice. Collectively, these results indicate that OASL1 deficiency suppresses the development of MS-like autoimmunity and suggest that negative regulators of IFN-I could be good therapeutic targets for treating MS in humans. PMID:27297771

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

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective. PMID:25676153

  10. Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

    A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU. This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors. While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity. TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT

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

    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.

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

    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.

  13. The mRNA and protein expression of folylpolyglutamate synthetase in methotrexate enantiomer-resistant A549 cell lines%信息动态

    2011-01-01

    Objective To study the expression of folylpolyglutamate synthetase ( FPGS ) in methotrexate ( MTX ) enantiomer-resistant A549 cell lines [ L-( + )-MTX and D-( - )-MTX ]. Methods The expression of FPGS on genetic and protein level was determined by FQ-PCR and Western blot in lung cancer A549 cells, and MTX enantiomer-resistant A549 cells [ L-( + )-MTX and D-( - )-MTX ], with the concentration of drug resistance was 15 μmol/L. Results The genetic expression level of FPGS was ( 0.80 ± 0. 09 ) and ( 2. 04 ± 0. 34 ) folds in L-( + )- MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells, there was statistical difference between two groups ( P < 0.05 ). The protein expression level of FPGS was ( 0. 85 ± 0. 12 ) and( 1.62 ± 0. 24 ) folds in L-( + )-MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells,there was statistical difference ( P < 0. 05 ). Conclusion The expression level of FPGS on genetic and protein level in drug resistant cells have been changed, and significant difference in two enantiomer-resistant cells are appeared.

  14. Effects of detergents on retinyl ester synthetase and all-trans:11-CIS retinoid isomerase activities in homogenates of bovine retinal pigment epithelium

    [11,12-3H] all-trans Retinol and various detergents were added to homogenates of fresh bovine retinal pigment epithelium. After dark incubation for 40 minutes at 37 degrees C, the retinoids were extracted and analyzed by a high resolution HPLC method. The detergents showed different effects on the retinyl ester synthetase (RES) and all-trans:11 cis retinoid isomerase (RI) activities. The detergent CHAPS (0.3%) almost totally destroyed RI activity without reducing RES activity. The same concentration of sodium dodecyl sulfate and Nonidet P-40 significantly reduced RES activity and totally destroyed RI activity. RES and RI activities were unaffected by 0.3% Mega 8, a nonionic detergent, but were inhibited by 1% Mega 8. Thus, because of these differential effects of detergents, RES and RI probably are different enzymes rather than a single multifunctional enzyme. Because isomerization was always inhibited more than esterification, our findings also accord with the esterification/isomerization mechanism recently reported by Rando et al

  15. Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein: A potential for developing hepatitis C virus targeting gene therapy

    Ying Wang; Shan-Shan Mao; Qiong-Qiong He; Yuan Zi; Ji-Fang Wen; De-Yun Feng

    2009-01-01

    AIM: To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed.RESULTS: L02/core cell line stably expressing HCV-core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

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

    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. 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

    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.

  18. Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

    Jiang, Yanfang; Morgan-Kiss, Rachael M.; Campbell, John W.; Chan, Chi Ho; Cronan, John E.

    2010-01-01

    Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously-supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function. PMID:20028080

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

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

    2015-06-01

    Here, we investigated the roles of YAL1 (FAA1) and FAT1 encoding acyl-CoA synthetases (ACSs) and three additional orthologs of ACS genes FAT2-FAT4 of the yeast Yarrowia lipolytica in the assimilation or utilization of n-alkanes and fatty acids. ACS deletion mutants were generated to characterize their function. The FAT1 deletion mutant exhibited decreased growth on n-alkanes of 10-18 carbons, whereas the FAA1 mutant showed growth reduction on n-alkane of 16 carbons. However, FAT2-FAT4 deletion mutants did not show any growth defects, suggesting that FAT1 and FAA1 are involved in the activation of fatty acids produced during the metabolism of n-alkanes. In contrast, deletions of FAA1 and FAT1-FAT4 conferred no defect in growth on fatty acids. The wild-type strain grew in the presence of cerulenin, an inhibitor of fatty acid synthesis, by utilizing exogenously added fatty acid or fatty acid derived from n-alkane when oleic acid or n-alkane of 18 carbons was supplemented. However, the FAA1 deletion mutant did not grow, indicating a critical role for FAA1 in the utilization of fatty acids. Fluorescent microscopic observation and biochemical analyses suggested that Fat1p is present in the peroxisome and Faa1p is localized in the cytosol and to membranes. PMID:26019148

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

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

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification. PMID:26882849

  1. Studies on the Rice LEAF INCLINATION1 (LC1),an IAA-amido Synthetase, Reveal the Effects of Auxin in Leaf Inclination Control

    Shu-Qing Zhao; Jing-Jing Xiang; Hong-Wei Xue

    2013-01-01

    The angle of rice leaf inclination is an important agronomic trait and closely related to the yields and architecture of crops.Although few mutants with altered leaf angles have been reported,the molecular mechanism remains to be elucidated,especially whether hormones are involved in this process.Through genetic screening,a rice gain-offunction mutant leaf inclination1,Ic1-D,was identified from the Shanghai T-DNA Insertion Population (SHIP).Phenotypic analysis confirmed the exaggerated leaf angles of Ic1-D due to the stimulated cell elongation at the lamina joint.LC1 is transcribed in various tissues and encodes OsGH3-1,an indole-3-acetic acid (IAA) amido synthetase,whose homolog of Arabidopsis functions in maintaining the auxin homeostasis by conjugating excess IAA to various amino acids.Indeed,recombinant LC1 can catalyze the conjugation of IAA to Ala,Asp,and Asn in vitro,which is consistent with the decreased free IAA amount in Ic1-D mutant.Ic1-D is insensitive to IAA and hypersensitive to exogenous BR,in agreement with the microarray analysis that reveals the altered transcriptions of genes involved in auxin signaling and BR biosynthesis.These results indicate the crucial roles of auxin homeostasis in the leaf inclination control.

  2. Aminoacetylation Reaction Catalyzed by Leucyl-tRNA Synthetase Operates via a Self-Assisted Mechanism Using a Conserved Residue and the Aminoacyl Substrate.

    Aleksandrov, Alexey; Palencia, Andrés; Cusack, Stephen; Field, Martin

    2016-05-19

    Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors-namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein-and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction. PMID:27115861

  3. Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain.

    Franco, Tathyana Mar A; Rostirolla, Diana C; Ducati, Rodrigo G; Lorenzini, Daniel M; Basso, Luiz A; Santos, Diógenes S

    2012-01-01

    Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family. PMID:22119138

  4. A futile cycle, formed between two ATP-dependant -glutamyl cycle enzymes, -glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    Akhilesh Kumar; Anand Kumar Bachhawat

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the -glutamyl cycle leads to ATP depletion. The enzyme -glutamyl cysteine synthetase (-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, -glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  5. A facile reproducible radioimmunoassay of the mixed metabolites of prostaglandins E, suitable for measurement of relative differences of phospholipase/prostaglandin synthetase activity in vivo.

    Fretland, D J; Cammarata, P S

    1984-04-01

    A relatively simple, reproducible, radioimmunoassay for the mixed metabolites of prostaglandins E (U-PGE-M) in rat and human urine is described. Results of the assay of treated versus control urine extracts correlate well with differences expected from treatments known to alter in vivo phospholipase/prostaglandin synthetase activity. Cross-reactivity of heterogeneous metabolite antiserum with 5 available endogenous prostaglandins and a single metabolite was determined and showed little or no cross reaction. Sensitivity, within-assay precision, interassay reproducibility, and parallelism were also determined and found acceptable. Excretion rates of U-PGE-M by rats and humans were determined, and statistically significant differences could be shown, although absolute values were smaller than estimated absolute values obtained from mass-spectrometric measurements of single, purified metabolites. Normal human male excretion rates differed significantly from those of females. Injection of prostaglandin E1 caused a significant rise in U-PGE-M excretion in rats whereas aspirin and indomethacin caused it to fall. U-PGE-M excretion rates of spontaneous hypertensive rats were significantly less than rates of normotensive controls. Adrenalectomy resulted in excretion of significantly larger amounts of U-PGE-M than in normal or sham-operated controls. A screen of clinically active pharmacological agents and hormones gave results consistent with previously published reports. PMID:6427792

  6. The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin.

    Amy J Gerc

    Full Text Available There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS assembly line (Alb4/5/6, tailoring enzymes (Alb2/3 and an export/resistance protein (Alb1, and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.

  7. Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

    Esmaeel, Qassim; Pupin, Maude; Kieu, Nam Phuong; Chataigné, Gabrielle; Béchet, Max; Deravel, Jovana; Krier, François; Höfte, Monica; Jacques, Philippe; Leclère, Valérie

    2016-06-01

    Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains. PMID:27060604

  8. 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

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

    2012-01-01

    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 gh3.5-1Dlpad3-1,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.

  9. 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?

    Verkhratsky Alexei

    2011-07-01

    Full Text Available Abstract Astrocytes are fundamental for brain homeostasis and the progression and outcome of many neuropathologies including Alzheimer's disease (AD. In the triple transgenic mouse model of AD (3xTg-AD generalised hippocampal astroglia atrophy precedes a restricted and specific β-amyloid (Aβ plaque-related astrogliosis. Astrocytes are critical for CNS glutamatergic transmission being the principal elements of glutamate homeostasis through maintaining its synthesis, uptake and turnover via glutamate-glutamine shuttle. Glutamine synthetase (GS, which is specifically expressed in astrocytes, forms glutamine by an ATP-dependent amination of glutamate. Here, we report changes in GS astrocytic expression in two major cognitive areas of the hippocampus (the dentate gyrus, DG and the CA1 in 3xTg-AD animals aged between 9 and 18 months. We found a significant reduction in Nv (number of cell/mm3 of GS immunoreactive (GS-IR astrocytes starting from 12 months (28.59% of age in the DG, and sustained at 18 months (31.65%. CA1 decrease of GS-positive astrocytes Nv (33.26% occurs at 18 months. This Nv reduction of GS-IR astrocytes is paralleled by a decrease in overall GS expression (determined by its optical density that becomes significant at 18 months (21.61% and 19.68% in DG and CA1, respectively. GS-IR Nv changes are directly associated with the presence of Aβ deposits showing a decrease of 47.92% as opposed to 23.47% in areas free of Aβ. These changes in GS containing astrocytes and GS-immunoreactivity indicate AD-related impairments of glutamate homeostatic system, at the advanced and late stages of the disease, which may affect the efficacy of glutamatergic transmission in the diseased brain that may contribute to the cognitive deficiency.

  10. Hereditary non-spherocytic haemolytic anaemia due to red blood cell glutathione synthetase deficiency in four unrelated patients from Spain: clinical and molecular studies.

    Corrons, J L; Alvarez, R; Pujades, A; Zarza, R; Oliva, E; Lasheras, G; Callis, M; Ribes, A; Gelbart, T; Beutler, E

    2001-02-01

    In four unrelated patients with chronic haemolysis and markedly reduced red blood cell (RBC) glutathione (49.5%, 12.6%, 11.5% and 15% of the normal concentration respectively), a severe glutathione synthetase (GSH-S, EC 6.3.2.3) deficiency was found. One case exhibited a neonatal haemolytic anaemia associated with oxoprolinuria, but without neurological manifestations. The family study revealed GSH-S activity in both parents to be around half the normal level, a finding consistent with the presumed autosomal recessive mode of inheritance of this enzymopathy. Two cases exhibited a well-compensated haemolytic syndrome without anaemia or splenomegaly at steady state. One of these cases was diagnosed after an episode of acute haemolytic anaemia after fava bean ingestion. The remaining patient suffered from moderate to severe chronic non-spherocytic haemolytic anaemia and splenomegaly, and required occasional blood transfusion for a haemolytic crisis associated with drug ingestion. In this patient, the anaemia was corrected by splenectomy. In addition to GSH-S, a panel of 16 other RBC enzyme activities was also studied in all the patients. Hexokinase, aldolase, glucose-6-phosphate dehydrogenase and pyruvate kinase activities all increased; these increases were to be expected, given the rise in the number of circulating reticulocytes. In two patients, the incubation of RBCs with hydrogen peroxide revealed an enhanced production of malonyldialdehyde. DNA analysis showed a homozygous state for 656 A-->G mutation in patients 2 and 3. The GSH-S gene of patient 1, studied elsewhere, revealed an 808 T-->C. The GSH-S gene of patient 4 was not available for study. The present study demonstrates that GSH-S deficiency is also present in Spain and further supports the molecular and clinical heterogeneity of this enzymopathy PMID:11167850

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

    Gareth T Banks

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

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

    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.

  13. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

    Lovley Derek R

    2010-07-01

    Full Text Available Abstract Background Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. Results CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. Conclusions The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of

  14. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

    2016-08-10

    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. PMID:27288593

  15. Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

    A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNA/sup fMet/). Incubation of the affinity-labeling tRNA/sup fMet/ derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNA/sup fMet/, but not by noncognate tRNA/sup Phe/. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins

  16. Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4 and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells

    Julia Krammer, Margarete Digel, Friedrich Ehehalt, Wolfgang Stremmel, Joachim Füllekrug, Robert Ehehalt

    2011-01-01

    Full Text Available Background: Understanding the mechanisms of long chain fatty acid (LCFA uptake in hepatic cells is of high medical importance to treat and to prevent fatty liver disease (FLD. ACSs (Acyl-CoA synthetases are a family of enzymes that catalyze the esterification of fatty acids (FA with CoA. Recent studies suggest that ACS enzymes drive the uptake of LCFA indirectly by their enzymatic activity and could promote special metabolic pathways dependent on their localization.The only protein located at the plasma membrane which has consistently been shown to enhance FA uptake is CD36.Aims: The current study investigated whether ACSs and CD36 could regulate hepatic LCFA uptake.Methods and Results: FATP2 and FATP4 were both localized to the ER of HuH7 and HepG2 cells as shown by double immunofluorescence in comparison to marker proteins. ACSL1 was located at mitochondria in both cell lines. Overexpression of FATP2, FATP4 and ACSL1 highly increased ACS activity as well as the uptake of [3H]-oleic acid and fluorescent Bodipy-C12 (B12 fatty acid. Quantitative FACS analysis showed a correlation between ACS expression levels and B12 uptake. FATP2 had the highest effect on B12 uptake of all proteins tested. CD36 was mainly localized at the plasma membrane. Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.Conclusion: Uptake of LCFA into hepatoma cells can be regulated by the expression levels of intracellular enzymes. We propose that ACS enzymes drive FA uptake indirectly by esterification. Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD or steatohepatitis (NASH.

  17. Astrocytes and glutamate homoeostasis in Alzheimer's disease: a decrease in glutamine synthetase, but not in glutamate transporter-1, in the prefrontal cortex

    Alexei Verkhratsky

    2013-10-01

    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.

  18. FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

    Kamei, Yasutomi; Hattori, Maki; Hatazawa, Yukino; Kasahara, Tomomi; Kanou, Masanobu; Kanai, Sayaka; Yuan, Xunmei; Suganami, Takayoshi; Lamers, Wouter H; Kitamura, Tadahiro; Ogawa, Yoshihiro

    2014-09-15

    Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle. PMID:25074987

  19. Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

    Yang, Zhengrong W.; Tendian, Susan W.; Carson, W. Michael; Brouillette, Wayne J.; Delucas, Lawrence J.; Brouillette, Christie G.

    2004-01-01

    Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD+ synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme’s catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, Ka, equal to 5.6 × 106/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer ⇆ folded monomer ⇆ unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large Ka down to ~106/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented. PMID:14978314

  20. Efficacy of Rituximab in Refractory Inflammatory Myopathies Associated with Anti- Synthetase Auto-Antibodies: An Open-Label, Phase II Trial.

    Yves Allenbach

    Full Text Available Anti-synthetase syndrome (anti-SS is frequently associated with myositis and interstitial lung disease (ILD. We evaluated prospectively, in a multicenter, open-label, phase II study, the efficacy of rituximab on muscle and lung outcomes.Patients were enrolled if they were refractory to conventional treatments (prednisone and at least 2 immunosuppressants. They received 1 g of rituximab at D0, D15, and M6. The primary endpoint was muscular improvement based on manual muscular testing (MMT10, Kendall score in 10 muscles at M12. Secondary endpoints were normalization of creatine kinase (CK level, ILD improvement based on forced vital capacity and/or diffuse capacity for carbon monoxide, and number and/or doses of associated immunosuppressants.Twelve patients were enrolled, and 10 completed the study. Only 2 patients presented an improvement of at least 4 points on at least two muscle groups (primary end-point. Overall, seven patients had an increase of at least 4 points on MMT10. CK level decreased from 399 IU/L (range, 48-11,718 to 74.5 IU/L (range, 40-47,857. Corticosteroid doses decreased from 52.5 mg/d (range, 10-70 to 9 mg/d (range, 7-65 and six patients had a decrease in the burden of their associated immunosuppressants. At baseline, all 10 patients presented with ILD. At M12, improvement of ILD was observed in 5 out of the 10 patients, stabilization in 4, and worsening in 1.This pilot study of rituximab treatment in patients with refractory anti-SS provided data on evolution of muscular and pulmonary parameters. Rituximab should now be evaluated in a larger, controlled study for this homogenous group of patients.Clinicaltrials.gov NCT00774462.

  1. Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana.

    Zhu, Chenguang; Fan, Qianlan; Wang, Wei; Shen, Chunlei; Meng, Xiangzong; Tang, Yuanping; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2014-02-25

    The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency. PMID:24334123

  2. Kinetic evidence for half-of-the-sites reactivity in tRNA/sup Trp/ aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

    The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flowed method. The transfer of tryptophan to tRNA/sup Trp/ was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. The authors measured either the formation of a single labeled aminoacyl-tRNA/sup Trp/ per enzyme subunit or the turnover of labeled aminoacyl-tRNA/sup Trp/ synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute half-of-the-sites reactivity); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNA/sup Trp/ under each labeling conditions were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate if aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process

  3. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

    The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH2 half, which is involved in the binding of the molecule of ATP that yields Pi, close to the regulatory domain

  4. 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.

    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.

  5. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    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. PMID:26575143

  6. Two distinct domains of the β subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection

    Zheng, Yong-Gang; Wei, Hui; Ling, Chen; Martin, Franck; Eriani, Gilbert; Wang, En-Duo

    2004-01-01

    The Aquifex aeolicus αβ-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the β subunit which is believed to bind tRNALeu. A yeast three-hybrid system was constructed on the basis of the interaction of the β subunit with its cognate tRNALeu. Then, seven mutated β subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle...

  7. Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase α-subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

    Man, Huimin; Pollmann, Stephan; Weiler, Elmar W.; Kirby, Edward G.

    2011-01-01

    The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript an...

  8. 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

    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 P212121, 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

  9. 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

    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.

  10. Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

    Barredo Julio C

    2006-05-01

    Full Text Available Abstract Background Expression of folylpoly-γ-glutamate synthetase (FPGS gene is two- to three-fold higher in B-precursor ALL (Bp- ALL than in T-lineage ALL (T-ALL and correlates with intracellular accumulation of methotrexate (MTX polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. Methods To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. Results FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH whereas in T-ALL (CCRF-CEM cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. Conclusion We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required

  11. Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

    Expression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate

  12. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( F). For longissimus dorsi, EAAC1 ( W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( 0.45) from omental or mesenchymal adipose. These data demonstrate (1) longissimus dorsi and adipose tissues of steers developing through typical production stages have different capacities for

  13. The multicenter study of a new assay for simultaneous detection of multiple anti-aminoacyl-tRNA synthetases in myositis and interstitial pneumonia.

    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.

  14. Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka

    Konradsen Flemming

    2007-03-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs in the Plasmodium vivax dihydrofolate reductase (Pfdhfr and dihydropteroate synthetase (Pvdhps genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP. Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ with primaquine (PQ and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka. Subjects and methods P. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs in an ELISA format. Results In the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1% blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%, the double mutant LRTS haplotype was the most frequently observed mutant (24.4% while the triple mutation (LRTN was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher

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

    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

  16. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  17. An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase† †Electronic supplementary information (ESI) available: General experimental procedures, primer sequences, and supporting figures. See DOI: 10.1039/c2cc34138g Click here for additional data file.

    Thibodeaux, Gabrielle N.; van der Donk, Wilfred A.

    2012-01-01

    Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

  18. Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

    Nussinov Ruth

    2006-03-01

    Full Text Available Abstract Background While the premise that lateral gene transfer (LGT is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS is found both as a class 1 and a class 2 enzyme (LysRS1-2. Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention. Results We applied a phylogenetic approach for determining the extent and origin of LGT in prokaryotic LysRS. Reconstructing species trees for Archaea and Bacteria, and inferring that their last common ancestors encoded LysRS1 and LysRS2, respectively, we studied the gains and losses of both classes. A complex pattern of LGT events emerged. In specific groups of organisms LysRS1 was replaced by LysRS2 (and vice versa. In one occasion, within the alpha proteobacteria, a LysRS2 to LysRS1 LGT was followed by reversal to LysRS2. After establishing the most likely LGT paths, we studied the possible origins of the laterally transferred genes. To this end, we reconstructed LysRS gene trees and evaluated the likely origins of the laterally transferred genes. While the sources of LysRS1 LGTs were readily identified, those for LysRS2 remain, for now, uncertain. The replacement of one LysRS by another apparently transits through a stage simultaneously coding for both synthetases, probably conferring a selective advantage to the affected organisms. Conclusion The family of LysRSs features complex LGT events. The currently available data were sufficient for identifying unambiguously the origins of LysRS1 but not of LysRS2 gene transfers. A selective advantage is suggested to organisms encoding simultaneously LysRS1-2.

  19. 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?

    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)

  20. The Effect of N-terminal Changes on Arginyl-tRNA Synthetase from Escherichia coli%N端的改变对大肠杆菌精氨酰-tRNA合成酶的影响

    刘文; 刘默芳; 夏宪; 王恩多; 王应睐

    2002-01-01

    得到了缺失Asn2的大肠杆菌(E.coli)精氨酰-tRNA合成酶(ArgRS)的变种和在其N端添加酵母ArgRS的N端23个氨基酸残基的嵌合变种. 它们的基因在大肠杆菌中表达时, 可能由于蛋白质误折叠, 大部分产生了包涵体. 与天然酶相比, 缺失变种保留了全部的氨基酸活化活力, 但氨基酰化活力下降了26%; 嵌合变种的以上两种活力下降了90%以上, 不能氨基酰化酵母tRNAArg. 缺失Asn2和Ile3的变种在E.coli中虽被表达, 但不稳定. 与天然酶相比, 嵌合变种的荧光光谱的最大发射波长向长波移动, 强度减小. 表明变种酶的构象和天然酶不同, 色氨酸更暴露. 用远紫外CD光谱预测变种酶的二级结构表明, 嵌合酶的α螺旋更少, β折叠更多, 无规卷曲稍多. E.coli ArgRS的N端结构域对活力和正确折叠是重要的.%An Asn2 deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn2 and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in amino acid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNAArg at all. The mutant deleted Asn2 and Ile3 was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in

  1. Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P

    GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, has been overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX–LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a = 54.80, b = 69.15, c = 94.08 Å, α = 95.47, β = 106.51, γ = 90.46°, and diffracted to 1.9 Å resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX–EF-P–LysAMS crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 105.93, b = 102.96, c = 119.94 Å, β = 99.4°, and diffracted to 2.5 Å resolution. Structure determination of the E. coli GenX–LysAMS and GenX–EF-P–LysAMS complexes by molecular replacement was successful and structure refinements are now in progress

  2. 大别山野生油茶中茶氨酸检测与茶氨酸合成酶基因克隆%Determination of theanine and cloning of theanine synthetase gene in Dabieshan wild Camellia oleifera

    马林龙; 顾辰辰; 邓威威; 金阳; 宛晓春

    2015-01-01

    茶氨酸是茶树叶片中最丰富的游离氨基酸,具有重要的生理药理功能,但迄今仅在蘑菇、蕈和一些山茶科(属)植物中检测到茶氨酸.茶氨酸因有一种独特的风味特色“umami鲜爽味”而被人类营养学广泛研究,并发现合成茶氨酸的植物不仅在植物分类上具有积极意义,而且对于植物资源的有效发掘有巨大经济价值;同时还可以间接去研究茶树中茶氨酸的代谢机理以及茶氨酸合成酶的分离纯化和 TS基因的克隆表达.该文运用 HPLC、LC-TOF/MS对大别山地区野生幼年与成年油茶根、叶中茶氨酸进行检测,并结合分子生物学手段对油茶中茶氨酸合成酶(theanine synthetase,TS)基因进行克隆与生物信息学分析.结果表明:在幼年的油茶根中检测到茶氨酸,含量为0.08 mg·g-1(鲜重),而在幼年的油茶叶片和成年油茶根、叶中均未检测到茶氨酸;在幼年油茶根中克隆出一条长为1071 bp油茶 TS 基因开放阅读框,其基因序列与茶树谷氨酰胺合成酶(glutamine synthetase,GS,AB117934)基因和TS(DD410896)序列的同源性达到98%,氨基酸序列与茶树中GS(AB117934)和TS(DD410896)的相似性高达99%.经生物信息学分析,该序列编码的TS蛋白具有20个磷酸化位点,不存在信号肽序列与跨膜结构,含有卷曲螺旋结构的亲水性细胞质蛋白.该研究将为油茶新经济价值的发掘,为茶氨酸在油茶中合成代谢途径的研究提供一定的理论基础,同时也为进一步研究茶氨酸在茶树中代谢机理提供了新的研究思路.%Theanine was discovered as the most abundant free amino acid in Camellia sinensis leaves with many physi-ological and pharmacological function.However,only limited reports have subsequently been published about theanine in the plant kingdom,for instance,mushroom,Xerocomus badius,and some theaceae plants.Theanine has been ex-tensively studied about human nutrition for tea unique taste

  3. 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

    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.

  4. 向日葵S-腺苷甲硫氨酸合成酶基因克隆与分析%Molecular Cloning and Bioinformatic Analysis of S-adenosylmethionine Synthetase Gene from Helianthus annuus

    周向红; 王萍

    2011-01-01

    To investigate contributions of S-adenosylmethionine synthetase ( SAMS) to tolerance to drought and salt in Helianthus annuus,we cloned one SAMS gene (designated as HaSAMSl ) using RT-PCR from H. Annus. The primers were designed according to results of computer-assisted cloning. Total RNAs were isolated from the leaves of H. Annum treated by salt. The HaSAMSl gene contained a complete coding sequence of 1173 bp,encoding the protein of 390 amino acids. HaSAMSl contained signature motifs of SAMS family, but had no membrane spanning domain and signal peptide. The HaSAMSl shared high amino acid sequence identity with the SAMSs from other organisms. The amino acid sequence of HaSAMSl showed 86. 8% - 96. 9% identity with the SAMSs from Chrysanthemum coronarium ,Arabidopsis thaliana and other higher plants. The cloning of HaSAMSl laid a foundation for further investigation of tolerance mechanism to drought and salt in H. Annuus.%为了研究S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)在向日葵(Helianthus annuus)抗旱和耐盐过程中的作用,先根据计算机辅助克隆结果设计引物,抽提盐胁迫向日葵叶片的总RNA,然后采用RT-PCR扩增技术克隆了向日葵的1个SAMS基因(命名为HaSAMS1),HaSAMS1基因的编码序列长1 173bp,编码390个氨基酸残基.HaSAMS1没有跨膜结构域,没有信号肽,含有SAMS蛋白的特征序列.HaSAMS1与其他物种的SAMS具有较高的序列相似性,与茼蒿(Chrysanthemum coronarium)和拟南芥(Arabidopsis thaliana)等高等植物SAMS的氨基酸序列一致性介于86.8% ~96.9%.HaSAMS1基因的克隆为进一步研究向日葵抗旱和耐盐机理奠定了基础.

  5. 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

    Conant Gavin C

    2010-07-01

    Full Text Available 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.

  6. Studies on the mutation breeding of γ-glutamylmethylamide synthetase-producing strain of Pseudomonas sp.and the optimum conditions for the production of γ-glutamylmethylamide synthetase%产γ-谷氨基甲酰胺合成酶菌株的筛选及产酶条件研究

    张亚丽; 李平; 问小强

    2011-01-01

    以假单胞茵(Pseudomonas sp.)为出发菌株,通过紫外诱变筛选得到一株γ-谷氨基甲酰胺合成酶高产菌株UV-19,其酶活提高32.54%.以突变株UV-19为供试菌株,对γ-谷氨基甲酰胺合成酶的发酵条件进行优化.首先利用Plackett-Burman设计筛选出影响较大的4个因素:葡萄糖、蛋白胨、起始pH值、装液量.在此基础上再利用CCD响应面分析法进行优化,得到最佳产酶培养条件为(g/L):葡萄糖15、蛋白胨12、NaCl 5.0、MgSO4·7H2O 0.2、K2HPO4·3H2O 0.5、甲胺盐酸盐1.0g/L、起始pH值6.5、装液量72mL/250mL.该优化条件下进行产酶培养,假单胞菌发酵产γ-谷氨基甲酰胺合成酶酶活力可达32.68U/mL.%Pseudomonas sp. was used as initial strain to isolate the high activity strain by means of mutation under the UV-light treatment. One strain UV-19 with higher enzymatic activity of γ-glutamylmethylamide synthetase increased by 32. 54% was obtained. The culture medium components and enzyme production conditions of mutant strain UV-19 were optimized by response surface methodology.Initially , Plackett-Burman design was used to evaluate the process variables which were relevant to the enzyme production. Four statistically significant parameters including glucose, peptone , initial pH and liquid volume were selected and utilized in order to optimize the process. The optimized levels of these factors were defined by response surface analysis ( RSA) . The optimized medium and conditions of producing enzyme were obtained. When the strain was cultured with the medium containing 15g/L glucose, 12 g/L peptone , 5g/L NaCl, 0. 2 g/L MgSO4 · 7H2O, 0. 5g/L K2 HPO4 · 3H2O, 1g/L CHsN · HCl, initial pH6. 5, enzyme activity was 32. 68 U/mL.

  7. 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.

    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±0.13μM, MIC of 3.53μM, 2.1log reduction against nutrient starved MTB, with 33% cytotoxicity at 50μM. It also showed 1.5log reduction of M. marinum load in Zebra fish at 10mg/kg. PMID:26867485

  8. Role of Key Residues at the Flavin Mononucleotide (FMN:Adenylyltransferase Catalytic Site of the Bifunctional Riboflavin Kinase/Flavin Adenine Dinucleotide (FAD  Synthetase from Corynebacterium ammoniagenes

    Susana Frago

    2012-11-01

    Full Text Available In mammals and in yeast the conversion of Riboflavin (RF into flavin mononucleotide (FMN and flavin adenine dinucleotide (FAD is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK and an ATP:FMN adenylyltransferase (FMNAT. However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS, which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS. Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase, and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

  9. Genome Sequence of Dickeya solani, a New soft Rot Pathogen of Potato, Suggests its Emergence May Be Related to a Novel Combination of Non-Ribosomal Peptide/Polyketide Synthetase Clusters

    Linda Garlant

    2013-12-01

    Full Text Available Soft rot Enterobacteria in the genera Pectobacterium and Dickeya cause rotting of many crop plants. A new Dickeya isolate has been suggested to form a separate species, given the name Dickeya solani. This bacterium is spreading fast and replacing the closely related, but less virulent, potato pathogens. The genome of D. solani isolate D s0432-1 shows highest similarity at the nucleotide level and in synteny to D. dadantii strain 3937, but it also contains three large polyketide/fatty acid/non-ribosomal peptide synthetase clusters that are not present in D. dadantii 3937. These gene clusters may be involved in the production of toxic secondary metabolites, such as oocydin and zeamine. Furthermore, the D. solani genome harbors several specific genes that are not present in other Dickeya and Pectobacterium species and that may confer advantages for adaptation to new environments. In conclusion, the fast spreading of D. solani may be related to the acquisition of new properties that affect its interaction with plants and other microbes in the potato ecosystem.

  10. Human cytomegalovirus-encoded miR-US4-1 promotes cell apoptosis and benefits discharge of infectious virus particles via down-regulation of glutaminyl-tRNA synthetase, QARS in HCMV-infected HELF cells

    Yaozhong Shao; Ying Qi; Yujing Huang; Zhongyang Liu; Yanping Ma; Xin Guo; Shujuan Jiang; Zhengrong Sun; Qiang Ruan

    2016-06-01

    Human cytomegalovirus (HCMV) can cause congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behaviour. Identification of functional target genes of miRNAs is an important step in studies on HCMV pathogenesis. In this study, Glutaminyl-tRNA Synthetase (QARS), which could regulate signal transduction pathways for cellular apoptosis, was identified as a direct target of hcmv-miR-US4-1. Apoptosis assay revealed that as silence of QARS by ectopic expression of hcmv-miR-US4-1 and specific small interference RNA of QARS can promote cell apoptosis in HCMV-infected HELF cells. Moreover, viral growth curve assays showed that hcmv-miR-US4-1 benefits the discharge of infectious virus particles. However, silence of hcmv-miR-US4-1 by its specific inhibitor overturned these effects. These results imply that hcmv-miR-US4-1 might have the same effects during HCMV nature infection. In general, hcmv-miR-US4-1 may involve in promoting cell apoptosis and benefiting discharge of infectious virus particles via down-regulation of QARS in HCMV-infected HELF cells.

  11. Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris%S-腺苷甲硫氨酸合成酶在毕赤酵母中的分泌表达

    王莲哲; 张现青; 李洋; 杨广笑; 何光源

    2009-01-01

    [Objective] The research aimed to study the secreted expression of S-adenosyl-L-methionine synthetase (SAMS) in Pichia pastoris. [Method] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sam2 was integrated into Pichia pastoris GS115 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-performance liquid chromatography (HPLC) by determining the production of S-adenosy-L-methionine (SAM) with the enzyme secreted. [Result] The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosylation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, especially the latter, after which the specific activity of SAMS was improved to 61.48 U/mg. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GS115 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

  12. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  13. 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

    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{sub 0}) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH{sub 0} induced behavior in E. coli and S. typhimurium by quantitation of H{sub 2} {sup 35}SO{sub 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{sub 0} 5.0 or shifted from growth at pH{sub 0} 7.0 to 5.0. Internal pH (pH{sub i}) changes occurring during steady state growth at low pH{sub 0}, and on shifting from pH{sub 0} 7.0 to 5.0, were measured using {sup 14}C-benzoic acid uptake.

  14. 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

    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 (pH0) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH0 induced behavior in E. coli and S. typhimurium by quantitation of H235SO4 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 pH0 5.0 or shifted from growth at pH0 7.0 to 5.0. Internal pH (pHi) changes occurring during steady state growth at low pH0, and on shifting from pH0 7.0 to 5.0, were measured using 14C-benzoic acid uptake

  15. 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

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

    2013-11-01

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

  16. 大强度游泳抑制小鼠骨骼肌谷氨酰胺合成酶活性%Inhibition of Glutamine Synthetase by High Intensity Swimming in Skeleton Muscle of Mice

    刘正冬; 李可基; 冯建英

    2001-01-01

    分析不同强度运动负荷对血浆和骨骼肌谷氨酰胺水平变化与骨骼肌谷氨酰胺合成酶活性变化,探讨运动影响血浆谷氨酰胺水平变化的机制。BAL/C小鼠不负重或2%负重不同强度游泳,每天2 h,持续8周。与对照组比较,大强度游泳组动物血浆(0.46±0.10、0.12±0.02 mmol/L,p<0.01)和肌肉(4.92±0.98、1.03±0.37 mmol/L,p<0.01)中的谷氨酰胺浓度降低,骨骼肌谷氨酰胺合成酶活性抑制(1874±191、1246±220 nmol/min/ g蛋白, p<0.01);而中等强度游泳升高血浆谷氨酰胺浓度(0.64±0.06 mmol/L,p<0.01)。小鼠在中等强度运动时,谷氨酰胺代谢表现了积极的代偿变化。但在大强度运动时,动物的体重增长减缓,血浆和肌肉谷氨酰胺水平均大幅度降低。结果提示,导致血浆谷氨酰胺水平降低的机制之一是骨骼肌中的谷氨酰胺合成酶抑制。%To analyze the effect of different intensity of exercise on glutamine levels in plasma and muscle and approach the mechanism of changes of plasma glutamine in exercise, BAL/C mice were randomly divided into middle intensity exercise (no extra load), high intensity exercise (2% bodyweight load) and control groups to swim for 2 hours/day and 8 weeks. Compared with the control group, high intensity exercise reduced glutamine levels in plasma (0.46±0.10, 0.12±0.02 mmol/L, p<0.01) and muscle (4.92±0.98, 1.03±0.37 mmol/L, p<0.01), and reduced the activity of glutamine synthetase in muscle (1874±191, 1246±220 mmol/min/mg, p

  17. 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

    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

  18. Characterization of a glutamine synthetase gene DvGS1 from Dunaliella viridis and investigation of the impact on expression of DvGS1 in transgenic Arabidopsis thaliana.

    Zhu, Chenguang; Fan, Qianlan; Wang, Wei; Shen, Chunlei; Wang, Peipei; Meng, Xiangzong; Tang, Yuanping; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2014-01-01

    A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency. PMID:24307252

  19. Circular dichroism and fluorescence spectroscopy of cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 demonstrates that group I cations are particularly effective in providing structure and stability to this halophilic protein.

    Christopher J Reed

    Full Text Available Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1, which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0-3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.

  20. 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

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent on the...... promoter in front of the gcaD gene. Analysis of cDNA molecules prepared with gcaD-prs-ctc-specified mRNA as the template revealed an RNA transcript that encompassed all three cistrons....