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Sample records for acid synthetase activity

  1. [Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid].

    PubMed

    Firsova, N A; Selivanova, K M; Alekseeva, L V; Evstigneeva, Z G

    1986-05-01

    The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.

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

    PubMed Central

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

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  3. Leucyl-tRNA synthetase activates Vps34 in amino acid-sensing mTORC1 signaling

    PubMed Central

    Yoon, Mee-Sup; Son, Kook; Arauz, Edwin; Han, Jung Min; Kim, Sunghoon; Chen, Jie

    2016-01-01

    SUMMARY Amino acid availability activates signaling by the mammalian target of rapamycin (mTOR) complex 1, mTORC1, a master regulator of cell growth. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. Here we identify leucyl-tRNA synthetase (LRS) as a leucine sensor for the activation of Vps34-PLD1 upstream of mTORC1. LRS is necessary for amino acid-induced Vps34 activation, cellular PI(3)P level increase, PLD1 activation, and PLD1 lysosomal translocation. Leucine binding but not tRNA charging activity of LRS is required for this regulation. Moreover, LRS physically interacts with Vps34 in amino acid-stimulatable non-autophagic complexes. Finally, purified LRS protein activates Vps34 kinase in vitro in a leucine-dependent manner. Collectively, our findings provide compelling evidence for a direct role of LRS in amino acid activation of Vps34 via a non-canonical mechanism, and fill a gap in the amino acid-sensing mTORC1 signaling network. PMID:27477288

  4. Intestinal acyl-CoA synthetase 5: activation of long chain fatty acids and behind.

    PubMed

    Klaus, Christina; Jeon, Min Kyung; Kaemmerer, Elke; Gassler, Nikolaus

    2013-11-14

    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. PMID:24259967

  5. Capsaicin, nonivamide and trans-pellitorine decrease free fatty acid uptake without TRPV1 activation and increase acetyl-coenzyme A synthetase activity in Caco-2 cells.

    PubMed

    Rohm, Barbara; Riedel, Annett; Ley, Jakob P; Widder, Sabine; Krammer, Gerhard E; Somoza, Veronika

    2015-01-01

    Red pepper and its major pungent component, capsaicin, have been associated with hypolipidemic effects in rats, although mechanistic studies on the effects of capsaicin and/or structurally related compounds on lipid metabolism are scarce. In this work, the effects of capsaicin and its structural analog nonivamide, the aliphatic alkamide trans-pellitorine and vanillin as the basic structural element of all vanilloids on the mechanisms of intestinal fatty acid uptake in differentiated intestinal Caco-2 cells were studied. Capsaicin and nonivamide were found to reduce fatty acid uptake, with IC₅₀ values of 0.49 μM and 1.08 μM, respectively. trans-Pellitorine was shown to reduce fatty acid uptake by 14.0±2.14% at 100 μM, whereas vanillin was not effective, indicating a pivotal role of the alkyl chain with the acid amide group in fatty acid uptake by Caco-2 cells. This effect was associated neither with the activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or the epithelial sodium channel (ENaC) nor with effects on paracellular transport or glucose uptake. However, acetyl-coenzyme A synthetase activity increased (p<0.05) in the presence of 10 μM capsaicin, nonivamide or trans-pellitorine, pointing to an increased fatty acid biosynthesis that might counteract the decreased fatty acid uptake.

  6. [Allosteric regulation of glucosamine synthetase activity by naphthoquinone derivatives and ethyl ester of di-(4-oxycumarinyl-3)-acetic acid].

    PubMed

    Sharaev, P N; Bogdanov, N G; Sarycheva, I K; Zhukova, E E

    1981-02-01

    The effects of derivatives of naphthoquinone, e.g. 2-methyl-3-phytyl-1,4-naphthoquinone (vitamin K1), 2-methyl-1,4-naphthoquinone (vitamin K3), 3-dihydro-2-methyl-1,4-naphthoquinone-2-sodium sulfonate (vicasol), derivatives of naphthohydroxyquinone, e.g. 2-methyl-1,4-naphthohydroxyquinone 1-monoacetate, 2-methyl-1,4-naphthohydroxyquinone 1,4-diacetate and the oxycumarine derivative di-(4-oxycumarinyl-3)-acetate ethyl ester (pelentan) on the activity of purified glutamine synthetase (EC 5.3.1.19) from rat liver were studied. The enzyme activity was increased under effects of vitamins K1 and K3 and was inhibited by pelentan. The data obtained are indicative of the allosteric effect of these compounds on the enzyme. PMID:7195738

  7. [Studies on regulation of glutamine synthetase activity from Streptomyces lincolnensis].

    PubMed

    Jin, Z; Jiao, R; Mao, Y

    2001-08-01

    Glutamine synthetase in crude extracts from Streptomyces lincolnensis growing under different nitrogen sources were studied. The results showed that NH4+ in high concentration repressed the biosynthesis of the enzyme. To determine whether Streptomyces lincolnensis has undergone covalent modification, a comparison of the glutamine synthetase isolated from cells grown on different nitrogen sources was made. No significant difference was observed in specific activity, pH optima, divalent cation response, and ultraviolet absorption spectra. Glutamine synthetase activity was not influenced by ammonia shock or snake venom phosphodiesterase treatment. Under these conditions, the activity of glutamine synthetase from K. aerogenes was markedly changed. There was therefore no evidence for enzymatic adenylylation of glutamine synthetase from Streptomyces lincolnensis. Glutamine synthetase was subject to feedback inhibition by end products of glutamine metabolism. Cumulative feedback inhibition of the Mn(2+)-dependent glutamine synthetase activity was demonstrated. These results suggest that glutamine synthetase from Streptomyces lincolnensis is an allosteric enzyme. PMID:12552916

  8. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    PubMed

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  11. [Regulation of glucosamine synthetase activity by cholesterol and hydrocortisone].

    PubMed

    Sharaev, P N; Ivanov, V G; Bogdanov, N G

    1988-09-01

    The effects of cholesterol and hydrocortisone (cortisol) on the activity of purified glucosamine synthetase from rat liver was studied in vitro. It was found that the enzyme activity is increased by cholesterol and inhibited by hydrocortisone. These steroids block the allosteric effect of vitamin K1 on the enzyme. There is evidence testifying to the allosteric type of cholesterol and hydrocortisone effects on glucosamine synthetase. PMID:3203113

  12. Effect of the non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activity toward medium-chain, long-chain and polyunsaturated fatty acids in mitochondria of mouse liver and brain.

    PubMed

    Kasuya, Fumiyo; Kazuhiro, Misumi; Tatsuya, Hasegawa; Nakamoto, Kazuo; Tokuyama, Shogo; Masuyama, Teiichi

    2013-02-01

    Effect of eleven non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activities toward octanoic, palmitic, arachidonic and docosahexaenoic acids was evaluated in mouse liver and brain mitochondria. The drugs tested were aspirin, salicylic acid, diflunisal, mefenamic acid, indomethacin, etodolac, ibuprofen, ketoprofen, naproxen, loxoprofen, flurbiprofen. In mouse liver mitochondria, diflunisal and mefenamic acid exhibited the inhibitory activities not only for octanoic acid (IC(50) = 78.7 and 64.7 µM) and but also for palmitic acid (IC(50) = 236.5 and 284.4 µM), respectively. Aspirin was an inhibitor for the activation of octanoic acid only (IC(50) = 411.0 µM). In the brain, mefenamic acid and diflunisal inhibited strongly palmitoyl-CoA formation (IC(50) = 57.3 and 114.0 µM), respectively. The activation of docosahexaenoic acid in brain was sensitive to inhibition by diflunisal and mefenamic acid compared with liver.

  13. Lipoic acid synthetase deficiency causes neonatal-onset epilepsy, defective mitochondrial energy metabolism, and glycine elevation.

    PubMed

    Mayr, Johannes A; Zimmermann, Franz A; Fauth, Christine; Bergheim, Christa; Meierhofer, David; Radmayr, Doris; Zschocke, Johannes; Koch, Johannes; Sperl, Wolfgang

    2011-12-01

    Lipoic acid is an essential prosthetic group of four mitochondrial enzymes involved in the oxidative decarboxylation of pyruvate, α-ketoglutarate, and branched chain amino acids and in the glycine cleavage. Lipoic acid is synthesized stepwise within mitochondria through a process that includes lipoic acid synthetase. We identified the homozygous mutation c.746G>A (p.Arg249His) in LIAS in an individual with neonatal-onset epilepsy, muscular hypotonia, lactic acidosis, and elevated glycine concentration in plasma and urine. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate and decreased pyruvate dehydrogenase complex activity. A pronounced reduction of the prosthetic group lipoamide was found in lipoylated proteins.

  14. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  15. Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

    PubMed Central

    McDonald, Roderick A.; Satyanarayana, T.; Kaplan, J. G.

    1973-01-01

    The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (Ki = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system. PMID:4698210

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

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

    PubMed

    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.

  20. Prostaglandin endoperoxide synthetase and the activation of benzo(a)pyrene to reactive metabolites in vivo in guinea pigs

    SciTech Connect

    Garattini, E.; Coccia, P.; Romano, M.; Jiritano, L.; Noseda, A.; Salmona, M.

    1984-11-01

    The role of prostaglandin endoperoxide synthetase in the in vivo activation of benzo(a)pyrene to reactive metabolites capable of interacting irreversibly with cellular macromolecules was studied in guinea pig liver, lung, kidney, spleen, small intestine, colon, and brain. DNA and protein covalent binding experiments were made after systemic administration of acetylsalicylic acid (200 mg/kg) followed by radiolabeled benzo(a)pyrene (4 microgram/kg). Results are compared with a control situation in which the prostaglandin endoperoxide synthetase inhibitor (acetylsalicylic acid) was not administered. No decrease in the level of DNA or protein benzo(a)pyrene-derived covalent binding was observed in any of the tissues studied.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  2. Characterization of the Suillus grevillei quinone synthetase GreA supports a nonribosomal code for aromatic α-keto acids.

    PubMed

    Wackler, Barbara; Lackner, Gerald; Chooi, Yit Heng; Hoffmeister, Dirk

    2012-08-13

    The gene greA was cloned from the genome of the basidiomycete Suillus grevillei. It encodes a monomodular natural product biosynthesis protein composed of three domains for adenylation, thiolation, and thioesterase and, hence, is reminiscent of a nonribosomal peptide synthetase (NRPS). GreA was biochemically characterized in vitro. It was identified as atromentin synthetase and therefore represents one of only a limited number of biochemically characterized NRPS-like enzymes which accept an aromatic α-keto acid. Specificity-conferring amino acid residues--collectively referred to as the nonribosomal code--were predicted for the primary sequence of the GreA adenylation domain and were an unprecedented combination for aromatic α-keto acids. Plausible support for this new code came from in silico simulation of the adenylation domain structure. According to the model, the predicted residues line the active site and, therefore, very likely contribute to substrate specificity.

  3. Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase.

    PubMed

    Abe, Tomoko; Hashimoto, Yoshiteru; Hosaka, Hideaki; Tomita-Yokotani, Kaori; Kobayashi, Michihiko

    2008-04-25

    Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.

  4. SOLUBLE HEPATIC δ-AMINOLEVULINIC ACID SYNTHETASE: END-PRODUCT INHIBITION OF THE PARTIALLY PURIFIED ENZYME*

    PubMed Central

    Scholnick, Perry L.; Hammaker, Lydia E.; Marver, Harvey S.

    1969-01-01

    The present study confirms the existence of hepatic δ-aminolevulinic acid synthetase in the cytosol of the liver, suggests that this enzyme may be in transit to the mitochondria, and defines some of the characteristics of the partially purified enzyme. The substrate and cofactor requirements are similar to those of mitochondrial δ-aminolevulinic acid synthetase. Heme strongly inhibits the partially purified enzyme. A number of proteins that bind heme block this inhibition, which explains previous failures to demonstrate heme inhibition in crude systems. End-product inhibition of δ-aminolevulinic acid synthetase in the mitochondria may play an important role in the regulation of heme biosynthesis in eukaryotic cells. PMID:5257968

  5. Leucyl-tRNA synthetase: double duty in amino acid sensing.

    PubMed

    Durán, Raúl V; Hall, Michael N

    2012-08-01

    The cellular response to amino acids is controlled at the molecular level by TORC1. While many of the elements that participate in TORC1 signaling are known, we still have no clear idea how cells sense amino acids. Two recent studies found that leucyl-tRNA synthetase (LRS) is a leucine sensor for TORC1, in both yeast and mammalian cells.

  6. Mutation affecting regulation of synthesis of acetohydroxy acid synthetase in Escherichia coli K-12.

    PubMed Central

    Jackson, J H; Henderson, E K

    1975-01-01

    Altered regulation of synthesis of acetohydroxy acid synthetase (AHAS) was previously reported in a mutant of Escherichia coli strain K-12. The mutant strain, growing in minimal medium, exhibits a partial growth limiatation and derepression of AHAS, owing to deficient synthesis of isoleucine. The genetic lesion (ilvE503) causing the isoleucine limitation was shown to cause derepression of a valine-sensitive AHAS activity. The derepression effect of the ilvE503 mutation upon synthesis of AHAS was conclusively demonstrated by introducing both the ilvE503 allele and an altered AHAS (ilv-521) into the same cell. Evidence is presented that suggests the presence of multiple genetic regions for synthesis and control of the valine-sensitive AHAS activity. PMID:1089632

  7. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed. Images Fig. 4. Fig. 7. PMID:597251

  8. Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase.

    PubMed

    Roy, Hervé; Ling, Jiqiang; Alfonzo, Juan; Ibba, Michael

    2005-11-18

    Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol. PMID:16162501

  9. Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

    PubMed

    Takita, Teisuke; Nakagoshi, Makoto; Inouye, Kuniyo; Tonomura, Ben'ichiro

    2003-01-24

    Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue. PMID:12507472

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

    PubMed

    Dalessandro, G; Northcote, D H

    1981-01-01

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

  11. Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase.

    PubMed

    Haese, A; Pieper, R; von Ostrowski, T; Zocher, R

    1994-10-14

    Enniatin synthetase catalyzes the biosynthesis of N-methylated cyclohexadepsipeptides. The 347 kDa enzyme is encoded by the esyn1 gene of Fusarium scirpi and contains two domains (EA and EB) homologous to each other and to regions of other microbial peptide synthetases. Parts of the esyn1 gene were subcloned in frame to a small lacZ gene portion of Escherichia coli expression vectors. Overproduced recombinant proteins showed a high tendency towards inclusion body formation and could be only partially dissolved in 8 M urea or 6 M guanidine hydrochloride. After renaturation, a 121 kDa recombinant protein representing the N-terminal conserved domain EA of enniatin synthetase was shown to activate D-hydroxyisolvaleric acid via adenylation. Similarly, a 158 kDa recombinant protein comprising the C-terminal conserved domain EB catalyzed the activation of the substrate amino acid (e.g. L-valine). Moreover, this protein could be photolabeled with S-[methyl-14C]adenosyl-L-methionine, (AdoMet) indicating the presence of the methyltransferase. Both functions, L-valine activation and AdoMet binding, could be assigned to a 108 kDa recombinant protein encompassing the A and the M segment of domain EB. The fact that a 65 kDa recombinant protein representing the M portion could be photolabeled, indicated the localization of the methyltransferase in this region. Three deletion mutants of the 65 kDa protein were shown to be inactive with respect to UV-induced AdoMet labeling. PMID:7932733

  12. Activity of formylphosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

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

    1986-05-01

    Formylphosphate (FP), a putative enzyme-bound intermediate in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase, was synthesized from formylfluoride and Pi. Measurement of hydrolysis rates by /sup 31/P NMR showed that FP is very unstable with a half-life of 48 min at 20/sup 0/C and pH 7. At pH 7 hydrolysis occurs with O-P bond cleavage as shown by /sup 18/O incorporation from /sup 18/O-H/sub 2/O into Pi. The substrate activity of FP was tested in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase isolated from Clostridium cylindrosporum. MgATP + H/sub 4/folate + HCOO/sup -/ in equilibrium MgADP + Pi +N/sup 10/-formylH/sub 4/folate FP supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formylH/sub 4/folate is produced from H/sub 4/-folate and FP but only if ADP is present, and ATP is produced from FP and ADP but only if H/sub 4/folate is present. The requirements for H/sub 4/folate in the synthesis of ATP from ADP and FP and for ADP in the synthesis of N/sup 10/-formylH/sub 4/folate from FP and H/sub 4/folate, are consistent with past kinetic and isotope exchange studies which showed that the reaction proceeds by a sequential mechanism and that all three substrates must be present for any reaction to occur.

  13. Induction of fatty acid synthetase and acetyl-CoA carboxylase by isolated rat liver cells.

    PubMed

    Porter, J W; Swenson, T L

    1983-01-01

    Current studies on the synthesis of long-chain fatty acids by isolated rat liver cells are largely concerned with the regulation of the activity of previously existing acetyl-CoA carboxylase and fatty acid synthetase, and with the regulation of the quantity of these enzymes. These studies have required the development of methods for obtaining high yields of viable hepatocytes that respond to hormonal treatment. Such methods have been developed over the past 10-15 years through the efforts of several laboratories. These studies have also required the development of a method to determine whether a change in the activity of an enzyme is due to a modification of preexisting enzyme or to a change in quantity of that enzyme. The most satisfactory method to use for such studies is immunotitration of enzyme activity. In recent years studies on the regulation of acetyl-CoA carboxylase have largely centered upon the effect of phosphorylation-dephosphorylation on the activity of this enzyme and whether glucagon inhibits the activity of this enzyme through this process. Much data from a number of laboratories have suggested that glucagon regulates the activity of this enzyme through phosphorylation-dephosphorylation. However, several of these studies involved the use of crude systems in which competing enzymes and substrates that can significantly interfere with acetyl-CoA carboxylase activity measurements were still present. Hence, a confirmation of these studies needs to be carried out under conditions in which the effects of competing enzymes and substrates are eliminated. Studies on changes in quantity of acetyl-CoA carboxylase and fatty acid synthetase have shown that these enzymes are induced by the fasting and refeeding of animals. They have also shown that insulin stimulates (10- to 30-fold) the induction of these enzymes. This induction appears to be due to a change in the quantity of translatable mRNA which may, in turn, be due to a change in the rate of

  14. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    PubMed Central

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  15. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  16. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  17. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    SciTech Connect

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

    1986-11-01

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

  18. Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

    PubMed

    Jeitner, Thomas M; Battaile, Kevin; Cooper, Arthur J L

    2015-12-01

    The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

  19. Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

    PubMed

    Boehlein, S K; Schuster, S M; Richards, N G

    1996-03-01

    Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

  20. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  1. Synthesis and in vitro/in vivo Evaluation of the Antitrypanosomal Activity of 3-Bromoacivicin, a Potent CTP Synthetase Inhibitor

    PubMed Central

    Conti, Paola; Pinto, Andrea; Wong, Pui E; Major, Louise L; Tamborini, Lucia; Iannuzzi, Maria C; De Micheli, Carlo; Barrett, Michael P; Smith, Terry K

    2011-01-01

    Abstract The first convenient synthesis of enantiomerically pure (αS,5S)-α-amino-3-bromo-4,5-dihydroisoxazol-5-yl acetic acid (3-bromoacivicin) is described. We demonstrate that 3-bromoacivicin is a CTP synthetase inhibitor three times as potent as its 3-chloro analogue, the natural antibiotic acivicin. Because CTP synthetase was suggested to be a potential drug target in African trypanosomes, the in vitro/in vivo antitrypanosomal activity of 3-bromoacivicin was assessed in comparison with acivicin. Beyond expectation, we observed a 12-fold enhancement in the in vitro antitrypanosomal activity, while toxicity against mammalian cells remained unaffected. Despite its good in vitro activity and selectivity, 3-bromoacivicin proved to be trypanostatic and failed to completely eradicate the infection when tested in vivo at its maximum tolerable dose. PMID:21275056

  2. Active site nanospace of aminoacyl tRNA synthetase: difference between the class I and class II synthetases.

    PubMed

    Dutta, Saheb; Choudhury, Kaberi; Banik, Sindrila Dutta; Nandi, Nilashis

    2014-03-01

    The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the

  3. Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

    PubMed Central

    Burbaum, J. J.; Schimmel, P.

    1992-01-01

    Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304356

  4. Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase.

    PubMed

    Donohue, T J; Bernlohr, R W

    1981-10-01

    The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM). PMID:6169702

  5. Incorporation of hydrogen atoms from deuterated water and stereospecifically deuterium-labeled nicotin amide nucleotides into fatty acids with the Escherichia coli fatty acid synthetase system.

    PubMed

    Saito, K; Kawaguchi, A; Okuda, S; Seyama, Y; Yamakawa, T

    1980-05-28

    The mechanism of hydrogen incorporation into fatty acids was investigated with intact Escherichia coli cells, a crude enzyme preparation and purified reductases of fatty acid synthetase system. The distributions of deuterium atoms incorporated into fatty acids from 2H2O and stereospecifically deuterium-labeled NADPH or NADH were determined by mass spectrometry. When E. coli was grown in 2H2O, almost every hydrogen atom of cellular fatty acids was incorporated from the medium. When fatty acids were synthesized from acetyl-CoA, malonyl-CoA and NADPH in the presence of a crude enzyme preparation of either E. coli or Bacillus subtilis, almost every hydrogen atom was also incorporated from the medium. In contrast to these results, purified beta-ketoacyl acyl carrier reductase directly transferred the HB hydrogen of NADPH to beta-ketoacyl acyl carrier protein, and purified enoyl acyl carrier protein reductase also transferred the HB hydrogen of NADPH and NADH directly to enoyl acyl carrier protein. In the crude enzyme preparation of E. coli, we found high activities which exchanged the HB hydrogen of NADPH with the deuterium of 2h2o. the conflicting results of the origin of hydrogen atoms of fatty acids mentioned above are explained by the presence of enzymes, which catalyzed the rapid exchange of NADPH with the deterium of 2H2O prior to the reaction of fatty acid synthetase. PMID:6990992

  6. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  7. Effect of estrogen administration on rat liver 2-5A synthetase activity.

    PubMed

    Smekens, M; Dumont, J E; Degeyter, A; Galand, P

    1986-08-01

    Interferon-induced 2-5A synthetase is also present in various cells and tissues in the absence of any interferon treatment. The activity of this enzyme, which synthesizes a series of oligoadenylates, ppp(A2'p)n5'A (collectively referred to as 2-5A), was previously shown to vary with the growth status of liver tissue i.e., it decreased before and during the peak of DNA synthesis activity induced in rat liver by a two third hepatectomy. In the course of studies aimed at testing the hypothesis that 2-5A synthetase activity might exert negative control on normal cell growth and multiplication, we show here that a treatment of ovariectomized rats with a single dose of estradiol-17beta (100 micrograms/100 g body weight) induced a transient increase in the [3H]thymidine labelling index in the liver after 24 h and markedly decreased the 2-5A synthetase activity. A time course study revealed that 2-5A synthetase activity started to decrease after 3 h, reaching a minimal value (10% of the control level) after 12 h, then slowly increased to come back to control level at 48 h. These results, together with our similar data on regenerating liver, suggest that low 2-5A synthetase activity is permissive for acquisition of proliferative 'competence' by G0 cells. PMID:3730433

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

    PubMed

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

    2012-04-13

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

  9. Diffuse glutamine synthetase overexpression restricted to areas of peliosis in a β-catenin-activated hepatocellular adenoma: a potential pitfall in glutamine synthetase interpretation.

    PubMed

    Berry, Ryan S; Gullapalli, Rama R; Wu, Jin; Morris, Katherine; Hanson, Joshua A

    2014-08-01

    Hepatocellular adenomas have recently been classified into four subtypes based on molecular findings: hepatocyte nuclear factor 1α (HNF1α) inactivated, inflammatory/telangiectatic, β-catenin activated, and unclassifiable. β-catenin-activated adenomas have the potential for malignant transformation and are thus important to recognize. Diffuse glutamine synthetase immunohistochemical positivity has been shown to be a reliable surrogate marker for β-catenin activation, though variations in staining patterns may be difficult to interpret. We report a case of a peliotic adenoma that was morphologically consistent with a β-catenin wild-type hepatocellular adenoma but harbored a β-catenin mutation by molecular analysis. The tumor lacked nuclear β-catenin positivity and demonstrated a hitherto undescribed pattern of glutamine synthetase overexpression restricted to areas of peliosis with mostly negative staining in non-peliotic areas. This pattern was initially interpreted as physiologic and may represent a potential pitfall in glutamine synthetase interpretation.

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

    PubMed Central

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

    2016-01-01

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

  11. Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

    A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low. Images PMID:2476665

  12. Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

    PubMed Central

    Fernandez, M P; Correa, J U; Cabib, E

    1982-01-01

    Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment. Images PMID:6216245

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

    DOEpatents

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

    2009-02-24

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

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

    DOEpatents

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

    2010-10-12

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

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

    SciTech Connect

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

    2012-02-14

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

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

    SciTech Connect

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

    2011-08-30

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

  17. Fat Metabolism in Higher Plants. XXXIV. Development of Fatty Acid Synthetase as a Function of Protein Synthesis in Aging Potato Tuber Slices

    PubMed Central

    Willemot, Claude; Stumpf, P. K.

    1967-01-01

    Experiments with inhibitors of protein synthesis (actinomycin D, puromycin, actidione) showed that the increase and the change in fatty acid synthetase activity, observed during the aging of potato disks, were accompanied by and related to a temporary rise in the rate of protein and RNA synthesis. These results concur with the earlier suggestion by Click and Hackett that the aging process involves a type of derepression. A possible course of events during aging, and possible derepression mechanisms are suggested and discussed. PMID:6045298

  18. Codon usage, amino acid usage, transfer RNA and amino-acyl-tRNA synthetases in Mimiviruses.

    PubMed

    Colson, Philippe; Fournous, Ghislain; Diene, Seydina M; Raoult, Didier

    2013-01-01

    Mimiviruses are giant viruses that infect phagocytic protists, including Acanthamoebae spp., which were discovered during the past decade. They are the current record holder among viruses for their large particle and genome sizes. One group is composed of three lineages, referred to as A, B and C, which include the vast majority of the Mimiviridae members. Cafeteria roenbergensis virus represents a second group, though the Mimiviridae family is still expanding. We analyzed the codon and amino acid usages in mimiviruses, as well as both the transfer RNA (tRNA) and amino acyl-tRNA synthetases. We confirmed that the codon and amino acid usages of these giant viruses are highly dissimilar to those in their amoebal host Acanthamoeba castellanii and are instead correlated with the high adenine and thymine (AT) content of Mimivirus genomes. We further describe that the set of tRNAs and amino acyl-tRNA synthetases in mimiviruses is globally not adapted to the codon and amino acid usages of these viruses. Notwithstanding, Leu(TAA)tRNA, present in several Mimivirus genomes and in multiple copies in some viral genomes, may complement the amoebal tRNA pool and may contribute to accommodate the viral AT-rich codons. In addition, we found that the genes most highly expressed at the beginning of the Mimivirus replicative cycle have a nucleotide content more adapted to the codon usage in A.castellanii.

  19. Effects of univalent cations on the activity of particulate starch synthetase.

    PubMed

    Nitsos, R E; Evans, H J

    1969-09-01

    An investigation was made to determine the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCl. Rubidium, cesium, and ammonium were 80% as effective as potassium while sodium and lithium were respectively 21% and 8% as effective as potassium. The K(A) for potassium was determined to be 6 mM. In the case of the particulate starch synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, considerable stimulation of enzyme activity was obtained by the addition of potassium to the reaction mixture. In these studies, low enzyme activity was observed in the absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been negative.

  20. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417]. PMID:23108

  1. Effect of treatment on erythrocyte phosphoribosyl pyrophosphate synthetase and glutathione reductase activity in patients with primary gout.

    PubMed Central

    Braven, J; Hardwell, T R; Hickling, P; Whittaker, M

    1986-01-01

    The activities of erythrocyte phosphoribosyl pyrophosphate (PRPP) synthetase and glutathione reductase (GTR) were studied in 26 patients with primary gout who were receiving no treatment or treatment with either allopurinol or azapropazone, and compared with the activity in a group of healthy controls. The activity of PRPP synthetase was significantly higher in the gout group and was not influenced by either drug. No significant difference in the activity of GTR was observed. The failure of either drug to suppress the increased activity of PRPP synthetase associated with gout is discussed. PMID:3024593

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

    PubMed Central

    Jiménez, Alberto; Santos, María A; Revuelta, José L

    2008-01-01

    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. PMID:18782443

  3. Regulation of Synthesis of the Branched-Chain Amino Acids and Cognate Aminoacyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli: a Common Regulatory Element

    PubMed Central

    Jackson, Julius; Williams, L. S.; Umbarger, H. E.

    1974-01-01

    Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli. PMID:4612020

  4. Inhibition of Long Chain Acyl Coenzyme A Synthetases during Fatty Acid Loading Induces Lipotoxicity in Macrophages

    PubMed Central

    Saraswathi, Viswanathan; Hasty, Alyssa H.

    2009-01-01

    OBJECTIVES Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs) which are independent risk factors for cardiovascular disease and diabetes. While impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. METHODS AND RESULTS Long chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the three ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels which induced lipotoxicity characterized by induction of apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high fat diet-fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. CONCLUSIONS Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages. PMID:19679826

  5. CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2010-11-01

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

  8. Sub-unit structure and specificity of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli

    PubMed Central

    Bruton, C. J.; Hartley, B. S.

    1968-01-01

    1. The purification of methionyl-transfer-RNA synthetase from Escherichia coli by a modified technique gives a 16% yield of a protein that appears homogeneous by the criteria of disc gel electrophoresis, ultracentrifugation and end-group analysis. 2. The molecular weight is 96000 and the protein consists of two sub-units of 48000, which appear to be identical. The amino acid composition and thiol content are reported. 3. Kinetic data are reported for analogues of methionine and for pure t-RNAF and t-RNAM, which are respectively the methionine transfer RNA that can exist in the formylmethionyl form and the one that can exist only in the methionyl form. The enzyme binds and acylates both species of transfer RNA identically. PMID:4874971

  9. Effect of some D-amino acids on the steady-state level of glutamine synthetase in Escherichia coli.

    PubMed

    Berberich, M A

    1985-09-01

    D-Glutamate can elicit an increase in the specific activity of glutamine synthetase (GS) when added to cells growing in the presence of high ammonia nitrogen. This effect is independent of glutamate dehydrogenase or glutamate synthase activities and could not be provoked by the addition of the various metabolites which participate in the regulation of GS in the covalent modification system. Neither could an increase in GS level be elicited by addition of any of the D-amino acids which function as allosteric effectors or inhibitors of GS activity. The increase in GS level could also be provoked by addition of D-lysine, D-threonine, or glycine to cells growing in an ammonia-rich medium. The increase in GS level generated by a mixture of D-glutamate, D-lysine, D-threonine, and glycine approximates the increase in GS level observed during step-down of a wild-type Escherichia coli culture from ammonia-sufficient to ammonia-limited growth conditions. Studies with mutants exhibiting alterations in GS regulation indicated that the increase elicited by the addition of D-amino acids depends on the presence of the wild-type glnD allele, although no direct correlation between a positive response and the state of adenylylation of GS can be made. PMID:2863253

  10. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

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

    1982-02-01

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

  11. [Cephalosporin-Acid Synthetase of Escherichia coli Strain VKPM B-10182: Genomic Context, Gene Identification, Producer Strain Production].

    PubMed

    Eldarov M, A; Sklyarenko, A V; Mardanov, A V; Beletsky, A V; Zhgun, A A; Dumina, M V; Medvedeva, N V; Satarova, D E; Ravin, N V; Yarockii, S V

    2015-01-01

    An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and β-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA. PMID:26596082

  12. Characterization of Cereulide Synthetase, a Toxin-Producing Macromolecular Machine

    PubMed Central

    Alonzo, Diego A.; Magarvey, Nathan A.; Schmeing, T. Martin

    2015-01-01

    Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride. PMID:26042597

  13. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

    PubMed Central

    Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

    2015-01-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  14. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase.

    PubMed

    Yao, Jiangwei; Dodson, V Joshua; Frank, Matthew W; Rock, Charles O

    2015-09-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  15. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  16. Human Tyr-tRNA synthetase is a potent PARP-1 activating effector target for resveratrol

    PubMed Central

    Sajish, Mathew; Schimmel, Paul

    2014-01-01

    Resveratrol (RSV) is reported to extend life span1,2 and provide cardio-neuro-protective3, anti-diabetic4, and anti-cancer effects3,5 by initiating a stress response2 that induces survival genes. Because human tyrosyl tRNA synthetase (TyrRS) translocates to the nucleus under stress conditions6, we considered the possibility that the tyrosine-like phenolic ring of RSV might fit into the active site pocket to effect a nuclear role. Here we present a 2.1Å co-crystal structure of RSV bound to the active site of TyrRS. RSV nullified the catalytic activity and redirected TyrRS to a nuclear function, stimulating NAD+-dependent auto-poly-ADP-ribosylation of PARP-1. Downstream activation of key stress signaling pathways were causally connected to TyrRS-PARP-1-NAD+ collaboration. This collaboration was also demonstrated in the mouse, and was specifically blocked in vivo by a RSV-displacing tyrosyl adenylate analog. In contrast to functionally diverse tRNA synthetase catalytic nulls created by alternative splicing events that ablate active sites7, here a non-spliced TyrRS catalytic null reveals a new PARP-1- and NAD+-dependent dimension to the physiological mechanism of RSV. PMID:25533949

  17. A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

    PubMed

    Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

    2015-05-01

    Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction. PMID:25626767

  18. A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

    PubMed

    Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

    2015-05-01

    Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction.

  19. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    SciTech Connect

    Safford, R.; de Silva, J.; Lucas, C.; Windust, J.H.C.; Shedden, J.; James, C.M.; Sidebottom, C.M.; Slabas, A.R.; Tombs, M.P.; Hughes, S.G.

    1987-03-10

    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 approx. 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.

  20. The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues.

    PubMed

    Taylor, J C; Markham, G D

    2000-02-11

    S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues. PMID:10660564

  1. Neddylation requires glycyl-tRNA synthetase to protect activated E2.

    PubMed

    Mo, Zhongying; Zhang, Qian; Liu, Ze; Lauer, Janelle; Shi, Yi; Sun, Litao; Griffin, Patrick R; Yang, Xiang-Lei

    2016-08-01

    Neddylation is a post-translational modification that controls the cell cycle and proliferation by conjugating the ubiquitin-like protein NEDD8 to specific targets. Here we report that glycyl-tRNA synthetase (GlyRS), an essential enzyme in protein synthesis, also plays a critical role in neddylation. In human cells, knockdown of GlyRS, but not knockdown of a different tRNA synthetase, decreased the global level of neddylation and caused cell-cycle abnormality. This function of GlyRS is achieved through direct interactions with multiple components of the neddylation pathway, including NEDD8, E1, and E2 (Ubc12). Using various structural and functional approaches, we show that GlyRS binds the APPBP1 subunit of E1 and captures and protects activated E2 (NEDD8-conjugated Ubc12) before the activated E2 reaches a downstream target. Therefore, GlyRS functions as a chaperone that critically supports neddylation. This function is probably conserved in all eukaryotic GlyRS enzymes and may contribute to the strong association of GlyRS with cancer progression. PMID:27348078

  2. The role of Drosophila cytidine monophosphate-sialic acid synthetase in the nervous system.

    PubMed

    Islam, Rafique; Nakamura, Michiko; Scott, Hilary; Repnikova, Elena; Carnahan, Mindy; Pandey, Dheeraj; Caster, Courtney; Khan, Saba; Zimmermann, Tina; Zoran, Mark J; Panin, Vladislav M

    2013-07-24

    While sialylation plays important functions in the nervous system, the complexity of glycosylation pathways and limitations of genetic approaches preclude the efficient analysis of these functions in mammalian organisms. Drosophila has recently emerged as a promising model for studying neural sialylation. Drosophila sialyltransferase, DSiaT, was shown to be involved in the regulation of neural transmission. However, the sialylation pathway was not investigated in Drosophila beyond the DSiaT-mediated step. Here we focused on the function of Drosophila cytidine monophosphate-sialic acid synthetase (CSAS), the enzyme providing a sugar donor for DSiaT. Our results revealed that the expression of CSAS is tightly regulated and restricted to the CNS throughout development and in adult flies. We generated CSAS mutants and analyzed their phenotypes using behavioral and physiological approaches. Our experiments demonstrated that mutant phenotypes of CSAS are similar to those of DSiaT, including decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects of neural transmission at neuromuscular junctions. Genetic interactions between CSAS, DSiaT, and voltage-gated channel genes paralytic and seizure were consistent with the hypothesis that CSAS and DSiaT function within the same pathway regulating neural excitability. Intriguingly, these interactions also suggested that CSAS and DSiaT have some additional, independent functions. Moreover, unlike its mammalian counterparts that work in the nucleus, Drosophila CSAS was found to be a glycoprotein-bearing N-glycans and predominantly localized in vivo to the Golgi compartment. Our work provides the first systematic analysis of in vivo functions of a eukaryotic CSAS gene and sheds light on evolutionary relationships among metazoan CSAS proteins.

  3. Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

    PubMed

    Cano, Florencia; Poderoso, Cecilia; Cornejo Maciel, Fabiana; Castilla, Rocío; Maloberti, Paula; Castillo, Fernanda; Neuman, Isabel; Paz, Cristina; Podestá, Ernesto J

    2006-06-01

    The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

  4. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

    PubMed

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just; Kelve, Merike; Uriz, Maria Jesus

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

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

    NASA Astrophysics Data System (ADS)

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

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

  6. Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?

    PubMed Central

    Bakken, A M; Farstad, M; Holmsen, H

    1991-01-01

    Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets. The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6). Other apparent Km values were very similar for all long-chain fatty acids tested. Palmitic acid inhibited the formation of [14C]arachidonoyl-CoA, and arachidonic acid inhibited the formation of [14C]palmitoyl-CoA, with [14C]arachidonate or [14C]palmitate respectively as substrate. After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks. After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with [14C]palmitate as substrate, and up to 116 nmol/min per mg of protein with [14C]arachidonate as substrate. The purification was respectively about 8- and 10-fold. The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory. PMID:1848073

  7. Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

    PubMed Central

    Donnelly, M. Lauren; Fimlaid, Kelly A.

    2016-01-01

    ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-03-18

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1990-05-01

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

  12. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

    PubMed

    Drozak, Jakub; Veiga-da-Cunha, Maria; Kadziolka, Beata; Van Schaftingen, Emile

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

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

    PubMed

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

    2015-11-23

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

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

    PubMed Central

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

    2015-01-01

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

  15. Acetoacetyl-CoA synthetase activity is controlled by a protein acetyltransferase with unique domain organization in Streptomyces lividans.

    PubMed

    Tucker, Alex C; Escalante-Semerena, Jorge C

    2013-01-01

    GCN5-type N-acetyltransferases (GNATs) are enzymes that catalyse the transfer of the acetyl group from acetyl-CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side-chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT-catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in Streptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify SlPatA, a GNAT in Streptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl-CoA synthetase (SlAacS). The latter has homologues in all domains of life. In vitro and in vivo evidence show that SlAacS is a bona fide acetoacetyl-CoA synthetase. SlPatA acetylates SlAacS more efficiently than it does acetyl-CoA synthetase, an enzyme known to be under acetylation control. SlPatA acetylates SlAacS at the active-site residue Lys617 and acetylation inactivates SlAacS. Acetylated SlAacS was deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl-CoA synthetase activity in all domains of life.

  16. Purification of isopenicillin N synthetase.

    PubMed Central

    Pang, C P; Chakravarti, B; Adlington, R M; Ting, H H; White, R L; Jayatilake, G S; Baldwin, J E; Abraham, E P

    1984-01-01

    Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus. PMID:6435606

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

    SciTech Connect

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

    1987-06-30

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

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

    SciTech Connect

    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.

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

    PubMed Central

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

    2014-01-01

    CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001 PMID:25030911

  20. Structural Analysis of the Active Site Geometry of N[superscript 5]-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli

    SciTech Connect

    Thoden, James B.; Holden, Hazel M.; Firestine, Steven M.

    2009-09-11

    N{sub 5}-Carboxyaminoimidazole ribonucleotide synthetase (N{sub 5}-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N{sub 5}-CAIR, MgADP, and P{sub i}. The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N{sub 5}-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P{sub i} bound in the active site cleft. These structures, determined to 1.6-{angstrom} resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P{sub i} complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P{sub i} rather than ATP. The bound P{sub i} shifts by approximately 3 {angstrom} relative to the {gamma}-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N{sub 5}-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N{sub 5}-CAIR synthetase.

  1. Effects of the chitin synthetase inhibitor plumbagin and its 2-demethyl derivative juglone on insect ecdysone 20-monooxygenase activity.

    PubMed

    Mitchell, M J; Smith, S L

    1988-12-01

    The chitin synthetase inhibitor plumbagin and its 2-demethyl derivative juglone were found to inhibit in a dose-response fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity associated with adult female Aedes aegypti, wandering stage larvae of Drosophila melanogaster, and fat body and midgut from last instar larvae of Manduca sexta. The concentration of these naphthoquinones required to elicit a 50% inhibition of the steroid hydroxylase activity in all the insects was approximately 1 x 10(-4) M.

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

    PubMed Central

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

    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 sham-operated 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. PMID:19533812

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

    PubMed

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

    2015-01-01

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

  4. A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity

    PubMed Central

    Huang, Qian; Zhou, Xiao-Long; Hu, Qin-Hua; Lei, Hui-Yan; Fang, Zhi-Peng; Yao, Peng; Wang, En-Duo

    2014-01-01

    Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNALeu. LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain–domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS. PMID:25051973

  5. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

    PubMed

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

    2015-12-01

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

  6. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP synthetase.

    PubMed

    De Clercq, E; Murase, J; Marquez, V E

    1991-06-15

    Cyclopentenylcytosine (Ce-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [herpes (cytomegalo), pox (vaccinia)], (+)RNA viruses [picorna (polio, Coxsackie, rhino), toga (Sindbis, Semliki forest), corona], (-)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis)] and (+/-)RNA viruses (reo). Ce-Cyd is a more potent antiviral agent than its saturated counterpart, cyclopentylcytosine (carbodine, C-Cyd). Ce-Cyd also has potent cytocidal activity against a number of tumor cell lines. The putative target enzyme for both the antiviral and antitumor action of Ce-Cyd is assumed to be the CTP synthetase that converts UTP to CTP. In keeping with this hypothesis was the finding that the antiviral and cytocidal effects of Ce-Cyd are readily reversed by Cyd and, to a lesser extent, Urd, but not by other nucleosides such as dThd or dCyd. In contrast, pyrazofurin and 6-azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases. PMID:1710119

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

    NASA Astrophysics Data System (ADS)

    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. Formation of a catalytically active complex between tRNAAsp and aspartyl-tRNA synthetase from yeast in high concentrations of ammonium sulphate.

    PubMed

    Giegé, R; Lorber, B; Ebel, J P; Moras, D; Thierry, J C; Jacrot, B; Zaccai, G

    1982-05-01

    The interactions of yeast tRNAAsp with cognate aspartyl-tRNA synthetase have been studied in high concentrations of either sodium chloride or ammonium sulphate by fluorescence titration and small-angle neutron scattering. In solutions containing more than 1M NaCl no complex is formed and enzymatic activity is abolished. In strong contrast, however, the physical measurements showed the formation of a two-to-one tRNA-enzyme complex, with high affinity, in 1.6 M (NH4)2SO4. Aminoacylation assays under the same salt conditions showed the enzymatic fixation of aspartic acid to tRNAAsp to occur at an appreciable rate. The present study emphasizes that the effects of salts on protein-nucleic acid interactions do not depend only on ionic strength but also on the nature of the salt. This study has allowed a rational approach to the crystallisation of a functional tRNAAsp-aspartyl-tRNA synthetase complex (Giegé, Lorber, Ebel, Thierry and Moras (1980) C.R. Acad. Sci. Paris, série D, 291, 393-396). PMID:7049254

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

    PubMed Central

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

    2016-01-01

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

  12. Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells.

    PubMed Central

    Pujade-Renaud, V.; Clement, A.; Perrot-Rechenmann, C.; Prevot, J. C.; Chrestin, H.; Jacob, J. L.; Guern, J.

    1994-01-01

    Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration. PMID:12232192

  13. Aminolaevulinate synthetase of Micrococcus denitrificans. Purification and properties of the enzyme, and the effect of growth conditions on the enzyme activity in cells.

    PubMed

    Tait, G H

    1973-02-01

    1. 5-Aminolaevulinate synthetase was detected in extracts of the non-photosynthetic bacterium Micrococcus denitrificans. 2. Activity is high in cells grown anaerobically in a defined nitrate medium, but is low in cells grown in an iron-deficient medium, and in cells grown aerobically. 3. Aminolaevulinate synthetase was purified extensively; it has a molecular weight of about 68000; apparent K(m) values for glycine, succinyl-CoA and pyridoxal phosphate are 12mm, 10mum and 11mum respectively; 2mum-haemin and 14mum-protoporphyrin inhibit by 50%. 4. The low activity of aminolaevulinate synthetase in iron-deficient cells increases on adding iron salts to cells only under conditions where protein synthesis can occur. 5. In defined nitrate medium with a high Ca(2+) concentration anaerobic growth yield is higher, but aminolaevulinate synthetase activity is lower than in cells grown in the medium with a low Ca(2+) concentration. In medium made from AnalaR constituents, growth yield is low and aminolaevulinate synthetase activity is high even in the presence of high concentrations of Ca(2+); on adding Cu(2+) (0.1mum) to the medium growth yield and aminolaevulinate synthetase activity become the same as in non-AnalaR medium. 6. Cells incubated under conditions where protein synthesis does not occur but where electron transport does, lose their aminolaevulinate synthetase activity rapidly. 7. The activities of aminolaevulinate dehydratase and succinic thiokinase do not change under any of the conditions of growth examined. 8. The possible mechanisms controlling aminolaevulinate synthetase activity and the role of this enzyme in controlling the synthesis of haem in this organism are discussed. PMID:4722442

  14. Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

    PubMed

    Päri, Mailis; Kuusksalu, Anne; Lopp, Annika; Kjaer, Karina Hansen; Justesen, Just; Kelve, Merike

    2014-02-01

    2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals.

  15. Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

    PubMed

    Päri, Mailis; Kuusksalu, Anne; Lopp, Annika; Kjaer, Karina Hansen; Justesen, Just; Kelve, Merike

    2014-02-01

    2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals. PMID:24184688

  16. Liver glutamine synthetase activity is markedly reduced in chronic ethanol-fed micropigs

    SciTech Connect

    Olin, K.L.; Zidenberg-Cherr, S.; Villanueva, J.; Halsted, C.H.; Keen, C.L. )

    1992-02-26

    The authors have reported that chronic ethanol (Et) feeding in the micropig results in changes in antioxidant defense including reductions in liver CuZnSOD and GSHPX activities, in vitamin E and A levels, and increases in liver MnSOD activity. Despite these alterations, liver mitochondria (mit) and microsome (mic) TBARS were lower in Et-fed than control (C) pigs. The significance of lower TBARS is unclear since the saturated to PUFA ratio was higher in mit and mic from Et than from C pigs. Thus in the current study they measured a non-lipid target of oxidative damage. Glutamine synthetase (GS) activity was measured as this protein is an excellent marker for oxidative damage due to the sensitivity of histidine residues to free radicals at the active site. Micropigs were fed high PUFA diets containing 40% of kcals as either Et or cornstarch (C) and 34% of kcals as corn oil. After 12 mo pigs were killed and livers removed. Fatty infiltration, inflammation and necrosis were observed in livers from Et pigs by 5 mo; collagen infiltration was apparent in 2 pigs by 12 mo. Et pigs had GS activities that were 90% lower than C pigs. The finding that liver Mn levels were higher in Et than in C pigs suggests that the low GS activity is not due to a reduction in Mn availability, although a shift in the distribution of Mn from GS to MnSOD may be involved. These data support the idea that chronic Et feeding is associated with oxidative damage and underscore the need to evaluate non-lipid targets as markers for oxidative damage.

  17. Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

    PubMed Central

    Andreev, Dmitri E.; Hirnet, Juliane; Terenin, Ilya M.; Dmitriev, Sergey E.; Niepmann, Michael; Shatsky, Ivan N.

    2012-01-01

    Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed. PMID:22373920

  18. Subependymal giant cell astrocytoma: a lesion with activated mTOR pathway and constant expression of glutamine synthetase.

    PubMed

    Buccoliero, Anna Maria; Caporalini, Chiara; Giordano, Flavio; Mussa, Federico; Scagnet, Mirko; Moscardi, Selene; Baroni, Gianna; Genitori, Lorenzo; Taddei, Gian Luigi

    2016-01-01

    Subependymal giant-cell astrocytoma (SEGA) is a rare tumor associated with tuberous sclerosis complex (TSC). TSC mainly involves the central nervous system (CNS) where SEGA, subependymal nodules, and cortical tubers may be present. First studies suggested the astrocytic nature of SEGA while successive studies demonstrated the mixed glio-neuronal nature. There are similarities between TSC-associated CNS lesions and type IIb focal cortical dysplasia (FCD). In all these pathologies, mammalian target of rapamycin (mTOR) pathway activation has been demonstrated. Recent data evidenced that balloon cells in FCD IIb express glutamine synthetase (GS). GS is involved in the clearance of glutamate. Cells expressing GS might exert an antiepileptic role. We evaluated by immunohistochemistry the glial fibrillary acidic protein (GFAP), neurofilaments (NF), and GS expression and the mTOR status (mTOR and phosphorylated ribosomal protein S6) in 16 SEGAs and 2 cortical tubers. Our purpose was to emphasize the mixed nature of SEGA and to further investigate the similarities between TSC-related CNS lesions (in particular SEGA) and FCD IIb. We confirm the glio-neuronal nature and the common activation of the mTOR pathway in SEGAs. In addition, we report for the first time that these tumors, analogously to FCD IIb, commonly express GS. Notably, the expression of mTOR, phosphorylated ribosomal protein S6, and GS was restricted to gemistocytic-like GFAP-negative cells. GS expression and mTOR pathway activation were also documented in cortical tubers. Further studies are necessary to understand the significance of GS expression in SEGAs as well as in cortical tubers. PMID:27390104

  19. Subependymal giant cell astrocytoma: a lesion with activated mTOR pathway and constant expression of glutamine synthetase.

    PubMed

    Buccoliero, Anna Maria; Caporalini, Chiara; Giordano, Flavio; Mussa, Federico; Scagnet, Mirko; Moscardi, Selene; Baroni, Gianna; Genitori, Lorenzo; Taddei, Gian Luigi

    2016-01-01

    Subependymal giant-cell astrocytoma (SEGA) is a rare tumor associated with tuberous sclerosis complex (TSC). TSC mainly involves the central nervous system (CNS) where SEGA, subependymal nodules, and cortical tubers may be present. First studies suggested the astrocytic nature of SEGA while successive studies demonstrated the mixed glio-neuronal nature. There are similarities between TSC-associated CNS lesions and type IIb focal cortical dysplasia (FCD). In all these pathologies, mammalian target of rapamycin (mTOR) pathway activation has been demonstrated. Recent data evidenced that balloon cells in FCD IIb express glutamine synthetase (GS). GS is involved in the clearance of glutamate. Cells expressing GS might exert an antiepileptic role. We evaluated by immunohistochemistry the glial fibrillary acidic protein (GFAP), neurofilaments (NF), and GS expression and the mTOR status (mTOR and phosphorylated ribosomal protein S6) in 16 SEGAs and 2 cortical tubers. Our purpose was to emphasize the mixed nature of SEGA and to further investigate the similarities between TSC-related CNS lesions (in particular SEGA) and FCD IIb. We confirm the glio-neuronal nature and the common activation of the mTOR pathway in SEGAs. In addition, we report for the first time that these tumors, analogously to FCD IIb, commonly express GS. Notably, the expression of mTOR, phosphorylated ribosomal protein S6, and GS was restricted to gemistocytic-like GFAP-negative cells. GS expression and mTOR pathway activation were also documented in cortical tubers. Further studies are necessary to understand the significance of GS expression in SEGAs as well as in cortical tubers.

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

    PubMed

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

    2016-05-01

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

  1. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    PubMed

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  2. 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

    SciTech Connect

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

    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

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

    SciTech Connect

    Hickey, E.W.; Hirshfield, I.

    1987-05-01

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

  4. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene1,2

    PubMed Central

    Xia, Mengna; Malkaram, Sridhar A.; Zempleni, Janos

    2013-01-01

    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines, and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites, and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2, and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon, and kidney cell lines; activities consistently followed the pattern P1> >P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene. PMID:24075901

  5. Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1.

    PubMed

    Sekulic, Nikolina; Dietrich, Kristen; Paarmann, Ingo; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2007-03-23

    Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.

  6. Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21.

    PubMed

    Barrientos, Álvaro; Merino, Estefanía; Casabon, Israël; Rodríguez, Joaquín; Crowe, Adam M; Holert, Johannes; Philipp, Bodo; Eltis, Lindsay D; Olivera, Elías R; Luengo, José M

    2015-01-01

    Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

  7. Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

    PubMed

    Padanad, Mahesh S; Konstantinidou, Georgia; Venkateswaran, Niranjan; Melegari, Margherita; Rindhe, Smita; Mitsche, Matthew; Yang, Chendong; Batten, Kimberly; Huffman, Kenneth E; Liu, Jingwen; Tang, Ximing; Rodriguez-Canales, Jaime; Kalhor, Neda; Shay, Jerry W; Minna, John D; McDonald, Jeffrey; Wistuba, Ignacio I; DeBerardinis, Ralph J; Scaglioni, Pier Paolo

    2016-08-01

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain. PMID:27477280

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

    PubMed

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

    2015-11-01

    Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1(T-/-)) are virtually unable to oxidize fatty acids, glucose use increases 8-fold to compensate. This metabolic switch activates mechanistic target of rapamycin complex 1 (mTORC1), which initiates growth by increasing protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, 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 and function, mTORC1 was inhibited by treating Acsl1(T-/-) and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1(T-/-) hearts, but did not improve ADP-stimulated oxygen consumption, which was likely caused by the 33-51% lower ATP synthase activity present in both vehicle- and rapamycin-treated 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 increasing the number of damaged mitochondria. PMID:26220174

  9. Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.

    PubMed

    Boehlein, S K; Nakatsu, T; Hiratake, J; Thirumoorthy, R; Stewart, J D; Richards, N G; Schuster, S M

    2001-09-18

    Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B. PMID:11551215

  10. A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

    PubMed Central

    Mareš, Jan; Hájek, Jan; Urajová, Petra; Kopecký, Jiří; Hrouzek, Pavel

    2014-01-01

    A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides. PMID:25369527

  11. Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis1

    PubMed Central

    Jessen, Dirk; Roth, Charlotte; Wiermer, Marcel

    2015-01-01

    In plants, fatty acids are synthesized within the plastid and need to be distributed to the different sites of lipid biosynthesis within the cell. Free fatty acids released from the plastid need to be converted to their corresponding coenzyme A thioesters to become metabolically available. This activation is mediated by long-chain acyl-coenzyme A synthetases (LACSs), which are encoded by a family of nine genes in Arabidopsis (Arabidopsis thaliana). So far, it has remained unclear which of the individual LACS activities are involved in making plastid-derived fatty acids available to cytoplasmic glycerolipid biosynthesis. Because of its unique localization at the outer envelope of plastids, LACS9 was regarded as a candidate for linking plastidial fatty export and cytoplasmic use. However, data presented in this study show that LACS9 is involved in fatty acid import into the plastid. The analyses of mutant lines revealed strongly overlapping functions of LACS4 and LACS9 in lipid trafficking from the endoplasmic reticulum to the plastid. In vivo labeling experiments with lacs4 lacs9 double mutants suggest strongly reduced synthesis of endoplasmic reticulum-derived lipid precursors, which are required for the biosynthesis of glycolipids in the plastids. In conjunction with this defect, double-mutant plants accumulate significant amounts of linoleic acid in leaf tissue. PMID:25540329

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

    SciTech Connect

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

    1987-02-01

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

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

    PubMed Central

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

    1987-01-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested. Images PMID:2433688

  14. Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II.

    PubMed

    Banerjei, L C; Davidson, J N

    1997-01-01

    Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation. PMID:9218000

  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.

    PubMed

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

    2013-11-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos.

    PubMed

    Mathur, M; Saluja, D; Sachar, R C

    1991-06-24

    About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature. PMID:1648405

  18. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

  19. Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2).

    PubMed

    Sevoz, C; Benoit, E; Buronfosse, T

    2000-04-01

    2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone. Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode. The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs). In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen. Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer. Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively. On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids. Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics.

  20. The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency.

    PubMed

    Taylor, John C; Takusagawa, Fusao; Markham, George D

    2002-07-30

    Crystallographic studies of Escherichia coli S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, MAT) have defined a flexible polypeptide loop that can gate access to the active site without contacting the substrates. The influence of the length and sequence of this active site loop on catalytic efficiency has been characterized in a mutant in which the E. coli MAT sequence (DRADPLEQ) has been replaced with the distinct sequence of the corresponding region of the otherwise highly homologous rat liver enzyme (HDLRNEEDV). Four additional mutants in which the entire DRADPLEQ sequence was replaced by five, six, seven, or eight glycines have been studied to unveil the effects of loop length and the influence of side chains. In all of the mutants, the maximal rate of S-adenosylmethionine formation (k(cat)) is diminished by more than 200-fold whereas the rate of hydrolysis of the tripolyphosphate intermediate is decreased by less than 3-fold. Thus, the function of the loop is localized to the first step in the overall reaction. The K(m) for methionine increases in all of the oligoglycine mutants, whereas the K(m) values for ATP are not substantially different. The k(cat) for the wild-type enzyme is decreased by increases in solution microviscosity with 55% of the maximal dependence. Thus, a diffusional event is coupled to the chemical step of AdoMet formation, which is known to be rate-limiting. The results indicate that a conformational change, possibly loop closure, is associated with AdoMet synthesis. The data integrate a previously discovered conformational change associated with PPP(i) binding to the E x AdoMet complex into the reaction sequence, reflecting a difference in protein conformation in the E x AdoMet x PPP(i) complex whether it is formed from the E x ATP x methionine complex or from binding of exogenous PPP(i). The temperature dependence of the k(cat) for S-adenosylmethionine formation shows that the removal of the side chains in the

  1. Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

    PubMed Central

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

    2015-01-01

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

  2. Peptide synthetase gene in Trichoderma virens.

    PubMed

    Wilhite, S E; Lumsden, R D; Straney, D C

    2001-11-01

    Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.

  3. Peptide Synthetase Gene in Trichoderma virens

    PubMed Central

    Wilhite, S. E.; Lumsden, R. D.; Straney, D. C.

    2001-01-01

    Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used. PMID:11679326

  4. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  5. Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica.

    PubMed

    Leisinger, Anne-Katrin; Janzen, Daniel H; Hallwachs, Winnie; Igloi, Gabor L

    2013-12-01

    L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of L-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256e258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring L-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall L-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the L-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents L-canavanine misincorporation into proteins. PMID:24446543

  6. Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees.

    PubMed

    Dalessandro, G; Northcote, D H

    1981-01-01

    The activity of a β-(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000-100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min(-1) mg(-1) of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min(-1) cell(-1)) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.

  7. Inhibitors of methionyl-tRNA synthetase have potent activity against Giardia intestinalis trophozoites.

    PubMed

    Ranade, Ranae M; Zhang, Zhongsheng; Gillespie, J Robert; Shibata, Sayaka; Verlinde, Christophe L M J; Hol, Wim G J; Fan, Erkang; Buckner, Frederick S

    2015-11-01

    The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs. PMID:26324270

  8. Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

    PubMed

    Yang, Chao; Liu, Fei; Chen, Shun; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

    2016-09-01

    2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study, we cloned and identified OASL in the Chinese goose for the first time. Goose 2'-5'-oligoadenylate synthetase-like (goOASL), including an ORF of 1527bp, encoding a protein of 508 amino acids. GoOASL protein contains 3 conserved motifs: nucleotidyltransferase (NTase) domain, 2'-5'-oligoadenylate synthetase (OAS) domain, and 2 ubiquitin-like (UBL) repeats. The tissue distribution profile of goOASL in 2-week-old gosling and adult goose were identified by Real-Time quantitative PCR, which revealed that the highest level of goOASL mRNA transcription was detected in the blood of adult goose and gosling. The mRNA transcription level of goOASL was upregulated in all tested tissues of duck Tembusu virus (DTMUV)-infected 3-day-old goslings, compared with control groups. Furthermore, using the stimulus Poly(I: C), ODN2006, R848, and lipopolysaccharide (LPS) as well as the viral pathogens DTMUV, H9N2 avian influenza virus (AIV), and gosling plague virus (GPV) to treat goose peripheral blood mononuclear cells (PBMCs) for 6 h, goOASL transcripts level was significantly upregulated in all treated groups. To further investigate the antiviral activity of goOASL, pcDNA3.1(+)-goOASL-His plasmid was constructed, and goOASL was expressed by the goose embryo fibroblast cells (GEFs) transfected with pcDNA3.1(+)-goOASL-His. Our research data suggested that Newcastle disease virus (NDV) replication (viral copies and viral titer) in GEFs was significantly reduced by the overexpression of goOASL protein. These data were meaningful for the antiviral immunity research of goose and shed light on the future prevention of NDV in fowl. PMID:27576097

  9. Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain.

    PubMed

    Richardson, Charles J; First, Eric A

    2016-03-15

    Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine. PMID:26890980

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

    PubMed

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

    1998-06-01

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

  11. Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme.

    PubMed

    Sheoran, Anita; First, Eric A

    2008-05-01

    The activation of D-tyrosine by tyrosyl-tRNA synthetase has been investigated using single and multiple turnover kinetic methods. In the presence of saturating concentrations of D-tyrosine, the activation reaction displays sigmoidal kinetics with respect to ATP concentration under single turnover conditions. In contrast, when the kinetics for the activation reaction are monitored using a steady-state (multiple turnover) pyrophosphate exchange assay, Michaelis-Menten kinetics are observed. Previous investigations indicated that activation of l-tyrosine by the K233A variant of Bacillus stearothermophilus tyrosyl-tRNA synthetase displays sigmoidal kinetics similar to those observed for activation of d-tyrosine by the wild-type enzyme. Kinetic analyses indicate that the sigmoidal behavior of the d-tyrosine activation reaction is not enhanced when Lys-233 is replaced by alanine. This supports the hypothesis that the mechanistic basis for the sigmoidal behavior is the same for both d-tyrosine activation by wild-type tyrosyl-tRNA synthetase and activation of l-tyrosine by the K233A variant. The observed sigmoidal behavior presents a paradox, as tyrosyl-tRNA synthetase displays an extreme form of negative cooperativity, known as "half-of-the-sites reactivity," with respect to tyrosine binding and tyrosyl-adenylate formation. We propose that the binding of D-tyrosine weakens the affinity with which ATP binds to the functional subunit in tyrosyl-tRNA synthetase. This allows ATP to bind initially to the nonfunctional subunit, inducing a conformational change in the enzyme that enhances the affinity of the functional subunit for ATP. The observation that sigmoidal kinetics are observed only under single turnover conditions suggests that this conformational change is stable over multiple rounds of catalysis. PMID:18319246

  12. The N Terminus of Pro-endothelial Monocyte-activating Polypeptide II (EMAP II) Regulates Its Binding with the C Terminus, Arginyl-tRNA Synthetase, and Neurofilament Light Protein*

    PubMed Central

    Xu, Haiming; Malinin, Nikolay L.; Awasthi, Niranjan; Schwarz, Roderich E.; Schwarz, Margaret A.

    2015-01-01

    Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1–70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147–312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1–70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1–70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases. PMID:25724651

  13. Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

    PubMed Central

    Becker, M A; Raivio, K O; Bakay, B; Adams, W B; Nyhan, W L

    1980-01-01

    An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates

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

    PubMed Central

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

    2016-01-01

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

  15. The 2'-5'-oligoadenylate synthetase in the lowest metazoa: isolation, cloning, expression and functional activity in the sponge Lubomirskia baicalensis.

    PubMed

    Schröder, Heinz C; Natalio, Filipe; Wiens, Matthias; Tahir, Muhammad Nawaz; Shukoor, Mohammed Ibrahim; Tremel, Wolfgang; Belikov, Sergey I; Krasko, Anatoli; Müller, Werner E G

    2008-02-01

    Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in a crude extract from L. baicalensis by application of a novel separation procedure based on polymer coated ferromagnetic nanoparticles. The particles were derivatized with a synthetic double-stranded RNA [dsRNA], synthetic poly(I:C), a known allosteric activator of the latent (2-5)A synthetase. These particles were used to separate a single 35kDa protein from a crude extract of L. baicalensis, which cross-reacted with antibodies raised against the sponge enzyme. In situ hybridization studies revealed that highest expression of the gene is seen in cells surrounding the aquiferous canals. Finally primmorphs, an in vitro cell culture system, from L. baicalensis were exposed to poly(I:C); they responded to this dsRNA with an increased expression of the (2-5)A synthetase gene already after a 1-day incubation period. We conclude that sponges contain the (2-5)A synthetase antiviral protection system.

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

    PubMed

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

    2015-12-11

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

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

    PubMed

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

    2015-01-01

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue. PMID:26658841

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

    PubMed

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

    2016-05-01

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

  19. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    PubMed

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  20. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

    PubMed Central

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

    2014-01-01

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

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

    PubMed

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

    2013-11-01

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

  2. FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

    PubMed

    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

  3. FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

    PubMed

    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.

  4. Conversion of UMP, an allosteric inhibitor of carbamyl phosphate synthetase, to an activator by modification of the UMP ribose moiety.

    PubMed

    Boettcher, B; Meister, A

    1981-06-25

    UMP is known to be an allosteric inhibitor of carbamyl phosphate synthetase, whereas IMP activates the enzyme. Surprisingly, dialdehyde UMP (prepared by periodate oxidation of UMP) was found to be a potent activator of the enzyme. Dialdehyde IMP, like IMP, produced activation. The corresponding dialcohol analogs of UMP and IMP (prepared by borohydride reduction of the dialdehyde analogs) had no effect on activity. These nucleotide interactions were further characterized by sedimentation velocity studies and by examination of the effects of inorganic phosphate on enzymatic activity. Although UMP promotes formation of an enzyme dimer, and IMP promotes formation of a tetramer (Powers, S. G., Meister, A., and Haschemeyer, R. H. (1980) J. Biol. Chem. 255, 1554-1558), the dialdehyde analogs of UMP and IMP both promote formation of mixed species. Low levels (less than 10 mM) of inorganic phosphate decrease the extent of activation by IMP, dialdehyde IMP, and dialdehyde UMP, but increase the extent of inhibition by UMP. The marked activation observed with dialdehyde UMP, and other considerations, suggest that the binding sites on the enzyme for IMP and UMP may overlap substantially. The findings also suggest that physiological levels of inorganic phosphate function in the modulation of the allosteric regulation of this enzyme by nucleotides. PMID:7240186

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

    PubMed Central

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

    1996-01-01

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

  6. [The activity of 2',5'-oligoadenylate-synthetase in rat spleen lymphocytes in the chronic ethanol intoxication and administration of zinc acetate].

    PubMed

    Chaĭka, V O; Kompanets', I V; Hadiliia, O P; Kharchenko, O I; Ostapchenko, L I

    2010-01-01

    It was shown that the activity of interferon-induced enzyme 2',5'-oligoadenylate-synthetase is suppressed in rat spleen lymphocytes under the chronic alcohol intoxication. The values of enzyme activity were minimal under the long-term action of etanol (21 day). The combined administration of zinc acetate and etanol to rats causes the increase of enzyme activity, the effect is most expressed on the late stages of alcohol intoxication development.

  7. Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

    PubMed

    Kugimiya, Akimitsu; Konishi, Hidenori; Fukada, Rie

    2016-03-01

    Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0).

  8. Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

    PubMed

    Kugimiya, Akimitsu; Konishi, Hidenori; Fukada, Rie

    2016-03-01

    Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0). PMID:26554858

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

    PubMed Central

    Berger, Stefanie; Welte, Cornelia; Deppenmeier, Uwe

    2012-01-01

    The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell. PMID:22927778

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

    SciTech Connect

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

    1996-05-15

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

  11. Effects of experimental hypo- and hyperthyroidism on hepatic long-chain fatty acyl-CoA synthetase and hydrolase.

    PubMed

    Dang, A Q; Faas, F H; Carter, W J

    1989-07-01

    The effects of T3 treatment and thyroidectomy on rat liver microsomal long-chain fatty acyl-CoA (LCFA-CoA) synthetase and LCFA-CoA hydrolase activities were determined. Hyperthyroid rats had a 36-42% decrease in LCFA-CoA synthetase with no change in hydrolase activity. This may contribute to the redirection of fatty acids from esterification to oxidation reactions in hyperthyroidism. Thyroidectomized rats had a 40-44% decrease in synthetase and a 27-42% decrease in LCFA-CoA hydrolase activity. The decrease in both LCFA-CoA synthetase and hydrolase activities in hypothyroidism may indicate that the LCFA-CoA turnover in this futile cycle is decreased in the liver.

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

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

  14. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

    NASA Technical Reports Server (NTRS)

    D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

    1987-01-01

    Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

  15. A Rationally Engineered Misacylating Aminoacyl-Trna Synthetase

    SciTech Connect

    Bullock, T.L.; Rodriguez-Hernandez, A.; Corigliano, E.M.; Perona, J.J.

    2009-05-12

    Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K{sub M} of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA{sup Gln} by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA{sup Gln} by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA{sup Gln} complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

  16. The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

    PubMed

    Cheng, Tai-Sheng

    2012-11-15

    The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2 mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H(2)O(2) and NBT stain for superoxide radicals (O(2)(·-)), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity.

  17. An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones.

    PubMed

    Cornejo Maciel, Fabiana; Maloberti, Paula; Neuman, Isabel; Cano, Florencia; Castilla, Rocío; Castillo, Fernanda; Paz, Cristina; Podestá, Ernesto J

    2005-06-01

    We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary

  18. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Δ ura8Δ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  19. The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.

    PubMed Central

    Kämper, U; Kück, U; Cherniack, A D; Lambowitz, A M

    1992-01-01

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast. Images PMID:1531084

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2011-01-01

    Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity. PMID:22140588

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

    PubMed

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

    2008-12-01

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

  3. The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity.

    PubMed

    Wright, Patricia; Anderson, Paul; Weng, Lei; Frick, Natasha; Wong, Wei Peng; Ip, Yuen Kwong

    2004-07-01

    The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small. PMID:15229866

  4. The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

    PubMed Central

    Fonné-Pfister, R; Chemla, P; Ward, E; Girardet, M; Kreuz, K E; Honzatko, R B; Fromm, H J; Schär, H P; Grütter, M G; Cowan-Jacob, S W

    1996-01-01

    (+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date. Images Fig. 4 Fig. 5 PMID:8790347

  5. 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.

    PubMed

    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

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

    SciTech Connect

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

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  7. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    SciTech Connect

    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.

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

    PubMed Central

    McCullough, Jerry L.; Maren, Thomas H.

    1973-01-01

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

  9. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis.

    PubMed

    Hattori, Y; Campbell, E B; Gross, S S

    1994-04-01

    Nitric oxide synthase produces NO, citrulline, water, and NADP at the expense of arginine, NADPH, and dioxygen. While citrulline has been considered to be an inert by-product of the high output inducible isoform of NO synthase (iNOS), we show here that immunostimulants induce a metabolic pathway in vascular smooth muscle cells, which enables them to regenerate arginine from citrulline. Regeneration of arginine from citrulline is accomplished by two urea cycle enzymes: arginino-succinate synthetase (AS) and argininosuccinate lyase (AL). Whereas AL is constitutive to vascular smooth muscle cells, AS mRNA and enzyme activity is markedly induced in cells by treatment with bacterial lipopolysaccharide (LPS). The induction of AS mRNA and activity by LPS follows a time course which mirrors that for iNOS but lags 1-2 h behind. As shown for iNOS, interferon-gamma does not itself induce AS but is synergistic with LPS. AS induction is suppressed by glucocorticoids, actinomycin D, and, to a lesser extent, cycloheximide. On the other hand, AS induction is unaffected by an excess of citrulline or the inhibitor of iNOS, N omega-methyl-L-arginine. Our results show the urea cycle enzymes AS and AL confer cells with the capacity to produce NO without a need for exogenous arginine. In conjunction with NOS, citric acid cycle enzymes that covert fumarate to oxaloacetate (fumarase and malate dehydrogenase) and oxaloacetate to aspartate (aspartate transaminase), AS and AL form a novel arginine-citrulline cycle that enables high output NO production by cells. PMID:7511585

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

    PubMed Central

    Pedersen, Carsten

    2016-01-01

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

  11. Sequence and Phylogenetic Analysis of FAD Synthetase

    NASA Astrophysics Data System (ADS)

    Schubert, Luisa; Frago, Susana; Martínez-Júlvez, Marta; Medina, Milagros

    2006-08-01

    An evolutionary analysis of the sequences available till now for FAD synthetases has been carried out. Several identical conserved residues have been observed along the sequences of all the FAD synthetases analyzed, which might correlate with role for these residues in the catalytic activity of the enzyme. Phylogenetic analysis shows that FAD synthetase sequences can be organized in two main clusters. One of them mainly contains temperature, pressure or pH resistant organisms, whereas in the other one organisms with pathogenic character can be found.

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

    PubMed Central

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

    2015-01-01

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

  13. Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.

    PubMed

    Van Dommelen, Anne; Spaepen, Stijn; Vanderleyden, Jozef

    2009-04-01

    Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2011-06-01

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

  16. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis.

    PubMed

    Dubey, Nidhi Chandrama; Tripathi, Bijay Prakash; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2015-01-28

    Acetyl coenzyme A (acetyl CoA) is an essential precursor molecule for synthesis of metabolites such as the polyketide-based drugs (tetracycline, mitharamycin, Zocor, etc.) fats, lipids, and cholesterol. Acetyl CoA synthetase (Acs) is one of the enzymes that catalyzes acetyl CoA synthesis, and this enzyme is essentially employed for continuous supply of the acetyl CoA for the production of these metabolites. To achieve reusable and a more robust entity of the enzyme, we carried out the immobilization of Acs on poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgels via adsorption. Cationic PNIPAm-PEI microgel was synthesized by one-step graft copolymerization of NIPAm and N,N-methylene bis-acrylamide (MBA) from PEI. Adsorption studies of Acs on microgel indicated high binding of enzymes, with a maximum binding capacity of 286 μg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of preformed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles. PMID:25561344

  17. 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.

    PubMed

    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 (2-6 μM), but different Km for arachidonic acid (0.5 and 6 μM 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

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

    PubMed

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

    1985-12-30

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

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

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

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

    PubMed

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

    1995-08-01

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

  1. Mammalian long-chain acyl-CoA synthetases.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2008-05-01

    Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

  2. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  3. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

    The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

  4. Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain.

    PubMed

    Rodriguez-Aparicio, L B; Guadalajara, A M; Rubio, V

    1989-04-01

    Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide. PMID:2742825

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

    PubMed

    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.

  6. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-04-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.

  7. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed Central

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-01-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover. Images PMID:6113590

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

    PubMed Central

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

    1995-01-01

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

  9. Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase.

    PubMed

    Hacker, C; Glinski, M; Hornbogen, T; Doller, A; Zocher, R

    2000-10-01

    N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases and S-adenosyl-l-methionine (AdoMet)-dependent N-methyltransferases. The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes. Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site. The role of four conserved motifs (I, (2085)VLEIGTGSGMIL; II/Y, (2105)SYVGLDPS; IV, (2152)DLVVFNSVVQYFTPPEYL; and V, (2194)ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied. Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding. Further shortening close to motif I resulted in loss of binding activity. Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity. Point mutations converting the conserved Tyr(223) (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to approximately 70%, indicating that Tyr(223) is important for AdoMet binding and that the aromatic Tyr(223) may be crucial for AdoMet binding in N-methylpeptide synthetases.

  10. Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase.

    PubMed

    Harnett, S P; Lowe, G; Tansley, G

    1985-06-01

    The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP. PMID:3893531

  11. Activation of PPARα by Fatty Acid Accumulation Enhances Fatty Acid Degradation and Sulfatide Synthesis.

    PubMed

    Yang, Yang; Feng, Yuyao; Zhang, Xiaowei; Nakajima, Takero; Tanaka, Naoki; Sugiyama, Eiko; Kamijo, Yuji; Aoyama, Toshifumi

    2016-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first reaction in the mitochondrial fatty acid β-oxidation pathway. VLCAD deficiency is associated with the accumulation of fat in multiple organs and tissues, which results in specific clinical features including cardiomyopathy, cardiomegaly, muscle weakness, and hepatic dysfunction in infants. We speculated that the abnormal fatty acid metabolism in VLCAD-deficient individuals might cause cell necrosis by fatty acid toxicity. The accumulation of fatty acids may activate peroxisome proliferator-activated receptor (PPAR), a master regulator of fatty acid metabolism and a potent nuclear receptor for free fatty acids. We examined six skin fibroblast lines, derived from VLCAD-deficient patients and identified fatty acid accumulation and PPARα activation in these cell lines. We then found that the expression levels of three enzymes involved in fatty acid degradation, including long-chain acyl-CoA synthetase (LACS), were increased in a PPARα-dependent manner. This increased expression of LACS might enhance the fatty acyl-CoA supply to fatty acid degradation and sulfatide synthesis pathways. In fact, the first and last reactions in the sulfatide synthesis pathway are regulated by PPARα. Therefore, we also measured the expression levels of enzymes involved in sulfatide metabolism and the regulation of cellular sulfatide content. The levels of these enzymes and cellular sulfatide content both increased in a PPARα-dependent manner. These results indicate that PPARα activation plays defensive and compensative roles by reducing cellular toxicity associated with fatty acids and sulfuric acid. PMID:27644403

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

    PubMed

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

    2016-10-01

    Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively. PMID:27405844

  13. Asparagine Synthesis in Pea Leaves, and the Occurrence of an Asparagine Synthetase Inhibitor 1

    PubMed Central

    Joy, Kenneth W.; Ireland, Robert J.; Lea, Peter J.

    1983-01-01

    Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [14C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [14C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine. Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue. PMID:16663168

  14. Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

    PubMed Central

    Eriani, G; Dirheimer, G; Gangloff, J

    1991-01-01

    The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged. Images PMID:2014166

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

    PubMed Central

    Fewer, David P; Rouhiainen, Leo; Jokela, Jouni; Wahlsten, Matti; Laakso, Kati; Wang, Hao; Sivonen, Kaarina

    2007-01-01

    Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. PMID:17908306

  16. [Changes in prostaglandin systhetase activity in mouse tissues as affected by S-[N-(3-aminopropyl)-2-aminoethyl] thiophosphoric acid].

    PubMed

    Prianishnikova, E N; Zhulanova, Z I; Romantsev, E F

    1980-01-01

    Effect of various concentrations of a radioprotector S-[N-(3 aminopropyl)-2-aminoethyl] thiophosphoric acid on the activity of prostaglandine synthetase was studied in mouse liver microsomes as well as in the soluble fractions of testicules and brain in vitro. The activity of prostaglandine synthetase was estimated by monitoring the formation of labelled PGF2 alpha and PGE2 from I-14C-arachidonic acid. The radioprotector at concentration 1.66 mg/ml stimulated formation of PGF2 alpha in all the tissues studied. At the lower concentrations of the radioprotector only slight stimulation of the biosynthesis of prostaglandines in testicules was noted. No effect on their synthesis in the brain soluble fraction could be observed while in the liver microsomes it was inhibited. The radioprotective substance studied apparently affected the cyclooxygenase activity, which is a key enzyme in the prostaglandine-synthesizing system.

  17. Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification.

    PubMed

    Nakano, Y; Itoh, M; Tanaka, E; Kimura, K

    1990-02-01

    Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.

  18. Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and mRNA in extrahepatic tissues of largemouth bass (Micropterus salmoides).

    PubMed

    Kong, H; Edberg, D D; Korte, J J; Salo, W L; Wright, P A; Anderson, P M

    1998-02-15

    Low levels of all of the enzymes required for urea synthesis via the urea cycle, including mitochondrial glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase III (CPSase III) and cytosolic glutamine synthetase, are known to be present in liver of the teleost fish largemouth bass (Micropterus salmoides). The levels of these enzymes are higher than those in most other teleosts, but they are significantly lower than the levels present in liver of ureoosmotic elasmobranchs. The purpose of this study was to assess the physiological role of CPSase III in the context of urea synthesis in adult bass. The results showed that urea-N accounts for about 30% of the total nitrogen (ammonia-N plus urea-N) excreted under control conditions. The rate of urea-N excretion did not increase in response to exposure to 1 mM NH4Cl (3 days) or 0.25 mM NH4Cl (12 days) in the external water, except for a transient increase after a day or two of exposure. CPSase III activity in liver also did not increase in response to exposure to ammonia. Adult largemouth bass, while apparently ureogenic, are primarily ammonotelic and remain so even in the presence of relatively high concentrations of ammonia in the external environment. The total units of CPSase III activity in liver are not sufficient to account for the quantity of urea that is excreted. However, CPSase III and ornithine carbamoyltransferase (OCTase) activities were found to be present in intestinal tissue and, unexpectedly, in muscle tissue. The total units of CPSase III and OCTase in muscle, intestine, and liver appear to be sufficient to account for the observed rate of urea excretion. The sequence of CPSase III cDNA was determined, which permitted the use of ribonuclease protection assays to demonstrate the presence of CPSase III mRNA in these tissues.

  19. Location of the binding site for the allosteric activator IMP in the COOH-terminal domain of Escherichia coli carbamyl phosphates synthetase.

    PubMed

    Bueso, J; Lusty, C J; Rubio, V

    1994-09-15

    Using UV-irradiation we cross-linked IMP, the allosteric activator of E. coli carbamyl phosphate synthetase (a heterodimer of 117.7 and 41.4 kDa subunits), to the large subunit of the enzyme. As in the native enzyme-IMP complex, the cross-linked complex was resistant to attack by trypsin. Thus, IMP is attached to its normal site and induces the normal conformational changes. Limited digestion of the [3H]IMP-labeled enzyme with V8 staphylococcal protease or with trypsin in the presence of SDS, and NH2-terminal sequencing, showed that [3H]IMP is cross-linked to the COOH-terminal 20 kDa domain of the large subunit, downstream of residue 912, supporting the proposal that this domain is specialized in effector binding and regulation. PMID:8093025

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

    SciTech Connect

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

    2011-08-19

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

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

    SciTech Connect

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara

    2012-08-31

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

  2. Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis.

    PubMed

    Bag, Satyabrata; Das, Bhabatosh; Dasgupta, Shreya; Bhadra, Rupak K

    2014-08-01

    Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

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

    PubMed

    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.

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

    PubMed

    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

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

    SciTech Connect

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  6. Mistranslation and its control by tRNA synthetases

    PubMed Central

    Schimmel, Paul

    2011-01-01

    Aminoacyl tRNA synthetases are ancient proteins that interpret the genetic material in all life forms. They are thought to have appeared during the transition from the RNA world to the theatre of proteins. During translation, they establish the rules of the genetic code, whereby each amino acid is attached to a tRNA that is cognate to the amino acid. Mistranslation occurs when an amino acid is attached to the wrong tRNA and subsequently is misplaced in a nascent protein. Mistranslation can be toxic to bacteria and mammalian cells, and can lead to heritable mutations. The great challenge for nature appears to be serine-for-alanine mistranslation, where even small amounts of this mistranslation cause severe neuropathologies in the mouse. To minimize serine-for-alanine mistranslation, powerful selective pressures developed to prevent mistranslation through a special editing activity imbedded within alanyl-tRNA synthetases (AlaRSs). However, serine-for-alanine mistranslation is so challenging that a separate, genome-encoded fragment of the editing domain of AlaRS is distributed throughout the Tree of Life to redundantly prevent serine-to-alanine mistranslation. Detailed X-ray structural and functional analysis shed light on why serine-for-alanine mistranslation is a universal problem, and on the selective pressures that engendered the appearance of AlaXps at the base of the Tree of Life. PMID:21930589

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

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-01-01

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

  8. Effects of detergents on retinyl ester synthetase and all-trans:11-CIS retinoid isomerase activities in homogenates of bovine retinal pigment epithelium

    SciTech Connect

    Shi, H.; Furr, H.C.; Olson, J.A. )

    1990-02-26

    (11,12-{sup 3}H) 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.

  9. Properties of Kaurene Synthetase from Marah macrocarpus1

    PubMed Central

    Frost, Russell G.; West, Charles A.

    1977-01-01

    synthetase activity A. Acetylcholine chloride and 2-chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A1, gibberellin A3, a mixture of gibberellins A4 and A7, gibberellin A13, and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm. PMID:16659781

  10. Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

    PubMed Central

    Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

    1999-01-01

    Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

  11. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity.

    PubMed

    Boniface, Audrey; Bouhss, Ahmed; Mengin-Lecreulx, Dominique; Blanot, Didier

    2006-06-01

    The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.

  12. Comparative Biochemical and Immunological Studies of Bacterial Glutamine Synthetases

    PubMed Central

    Tronick, Steven R.; Ciardi, Joseph E.; Stadtman, E. R.

    1973-01-01

    Antisera prepared against adenylylated and unadenylylated Escherichia coli glutamine synthetase cross-reacted with the glutamine synthetases from a number of gram-negative bacteria and one gram-variable species as demonstrated by immunodiffusion and inhibition of enzyme activity. In contrast, the antisera did not cross-react with the glutamine synthetases from gram-positive bacteria (with one exception) nor with the synthetases of higher organisms. Modification of the various glutamine synthetases by covalent attachment of adenosine 5′-monophosphate (or other nucleotides) was tested for by determining whether or not snake venom phosphodiesterase altered catalytic activity in a manner similar to its effect on adenylylated E. coli glutamine synthetase. Only the activity of the glutamine synthetases from gram-negative bacteria grown with specific levels of nitrogen sources could be altered by snake venom phosphodiesterase. In addition, a relative order of antigenic homology between cross-reacting enzymes was suggested based on the patterns of spur formation in the immunodiffusion assay. Images PMID:4125585

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

    PubMed

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

    2006-06-01

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

  14. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    MedlinePlus

    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 ...

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

    PubMed

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

    2016-06-01

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

  16. Evolution of lanthipeptide synthetases

    PubMed Central

    Zhang, Qi; Yu, Yi; Vélasquez, Juan E.; van der Donk, Wilfred A.

    2012-01-01

    Lanthionine-containing peptides (lanthipeptides) are a family of ribosomally synthesized and posttranslationally modified peptides containing (methyl)lanthionine residues. Here we present a phylogenomic study of the four currently known classes of lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). Although they possess very similar cyclase domains, class II–IV synthetases have evolved independently, and LanB and LanC enzymes appear to not always have coevolved. LanM enzymes from various phyla that have three cysteines ligated to a zinc ion (as opposed to the more common Cys-Cys-His ligand set) cluster together. Most importantly, the phylogenomic data suggest that for some scaffolds, the ring topology of the final lanthipeptides may be determined in part by the sequence of the precursor peptides and not just by the biosynthetic enzymes. This notion was supported by studies with two chimeric peptides, suggesting that the nisin and prochlorosin biosynthetic enzymes can produce the correct ring topologies of epilancin 15X and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial biosynthesis. Our study also demonstrates unexplored areas of sequence space that may be fruitful for genome mining. PMID:23071302

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. A facile reproducible radioimmunoassay of the mixed metabolites of prostaglandins E, suitable for measurement of relative differences of phospholipase/prostaglandin synthetase activity in vivo.

    PubMed

    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

  19. Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa

    PubMed Central

    Ackerley, David F.; Caradoc-Davies, Tom T.; Lamont, Iain L.

    2003-01-01

    Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. l-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including d-threonine, l-serine, and l-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases. PMID:12700264

  20. Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

    PubMed

    Knudsen, C T; Immerdal, L; Grunnet, N; Quistorff, B

    1992-02-15

    Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.

  1. Rotavirus Controls Activation of the 2′-5′-Oligoadenylate Synthetase/RNase L Pathway Using at Least Two Distinct Mechanisms

    PubMed Central

    Sánchez-Tacuba, Liliana; Rojas, Margarito; Arias, Carlos F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2′-5′ oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2′-5′-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2′-5′-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. IMPORTANCE Rotaviruses represent an important cause of severe gastroenteritis in the young of many animal species, including humans. In this work, we have found that the OAS/RNase L pathway is activated during rotavirus infection, but the virus uses two different strategies to prevent the

  2. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.

  3. Versatility of acyl-acyl carrier protein synthetases.

    PubMed

    Beld, Joris; Finzel, Kara; Burkart, Michael D

    2014-10-23

    The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms. PMID:25308274

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

    PubMed

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

    2010-01-21

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

  5. Functional Characterization of PyrG, an Unusual Nonribosomal Peptide Synthetase Module from the Pyridomycin Biosynthetic Pathway.

    PubMed

    Huang, Tingting; Li, Lili; Brock, Nelson L; Deng, Zixin; Lin, Shuangjun

    2016-08-01

    Pyridomycin is an antimycobacterial cyclodepsipeptide assembled by a nonribosomal peptide synthetase/polyketide synthase hybrid system. Analysis of its cluster revealed a nonribosomal peptide synthetase (NRPS) module, PyrG, that contains two tandem adenylation domains and a PKS-type ketoreductase domain. In this study, we biochemically validated that the second A domain recognizes and activates α-keto-β-methylvaleric acid (2-KVC) as the native substrate; the first A domain was not functional but might play a structural role. The KR domain catalyzed the reduction of the 2-KVC tethered to the peptidyl carrier protein of PyrG in the presence of the MbtH family protein, PyrH. PyrG was demonstrated to recognize many amino acids. This substrate promiscuity provides the potential to generate pyridomycin analogues with various enolic acids moiety; this is important for binding InhA, a critical enzyme for cell-wall biosynthesis in Mycobacterium tuberculosis.

  6. Genetic engineering activates biosynthesis of aromatic fumaric acid amides in the human pathogen Aspergillus fumigatus.

    PubMed

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H; Dahse, Hans-Martin; Brakhage, Axel A; Hoffmeister, Dirk

    2015-03-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

  7. Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H.; Dahse, Hans-Martin; Brakhage, Axel A.

    2014-01-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds. PMID:25527545

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

    PubMed Central

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

    1995-01-01

    Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia. PMID:7486915

  9. Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

    PubMed Central

    2012-01-01

    Background Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria. Results Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent. Conclusions Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene. PMID:22433663

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  12. Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways.

    PubMed

    Delannay, S; Charlier, D; Tricot, C; Villeret, V; Piérard, A; Stalon, V

    1999-03-01

    Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleotides and arginine biosynthetic pathways. The enzyme harbors a complex regulation, being activated by ornithine and inosine 5'-monophosphate (IMP), and inhibited by UMP. CPSase mutants obtained by in vivo mutagenesis and selected on the basis of particular phenotypes have been characterized kinetically. Two residues, serine 948 and threonine 1042, appear crucial for allosteric regulation of CPSase. When threonine 1042 is replaced by an isoleucine residue, the enzyme displays a greatly reduced activation by ornithine. The T1042I mutated enzyme is still sensitive to UMP and IMP, although the effects of both regulators are reduced. When serine 948 is replaced by phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent. When correlating these observations to the structural data recently reported, it becomes clear that both mutations, which are located in spatially distinct regions corresponding respectively to the ornithine and the UMP/IMP binding sites, have coupled effects on the enzyme regulation. These results provide an illustration that coupling of regulatory pathways occurs within the allosteric subunit of E. coli CPSase. In addition, other mutants have been characterized, which display altered affinities for the different CPSase substrates and also slightly modified properties towards the allosteric effectors: P165S, P170L, A182V, P360L, S743N, T800F and G824D. Kinetic properties of these modified enzymes are also presented here and correlated to the crystal structure of E. coli CPSase and to the phenotype of the mutants. PMID:10047492

  13. Dependence of in vivo glutamine synthetase activity on ammonia concentration in rat brain studied by 1H - 15N heteronuclear multiple-quantum coherence-transfer NMR.

    PubMed Central

    Kanamori, K; Ross, B D; Kuo, E L

    1995-01-01

    The dependence of the in vivo rate of glutamine synthesis on the substrate ammonia concentration was studied in rat brain by 1H-15N heteronuclear multiple-quantum coherence-transfer NMR in combination with biochemical techniques. In vivo rates were measured at various steady-state blood and brain ammonia concentrations within the ranges 0.4-0.55 mumol/g and 0.86-0.98 mumol/g respectively, after low-rate intravenous 15NH4+ infusion (isotope chase). The rate of glutamine synthesis at steady state was determined from the change in brain [5-15N]glutamine levels during isotope chase, observed selectively through the amide proton by NMR, and 15N enrichments of brain glutamine and of blood and brain ammonia measured byN gas chromatography-MS. The in vivo rate (v) was 3.3-4.5 mumol/h per g of brain at blood ammonia concentrations (s) of 0.40-0.55 mumol/g. A linear increase of 1/v with 1/s permitted estimation of the in vivo glutamine synthetase (GS) activity at a physiological blood ammonia concentration to be 0.4-2.1 mumol/h per g. The observed ammonia-dependence strongly suggests that, under physiological conditions, in vivo GS activity is kinetically limited by sub-optimal in situ concentrations of ammonia as well as glutamate and ATP. Comparison of the observed in vivo GS activity with the reported in vivo rates of glutaminase and of gamma-aminobutyrate (GABA) synthesis suggests that, under mildly hyperammonaemic conditions, glutamine is synthesized at a sufficiently high rate to serve as a precursor of GABA, but glutaminase-catalysed hydrolysis of glutamine is too slow to be the sole provider of glutamate used for GABA synthesis. PMID:7487913

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

    DOEpatents

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

    2008-04-08

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

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

    DOEpatents

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

    2012-05-22

    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.

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

    DOEpatents

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

    2011-09-06

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

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

    DOEpatents

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

    2010-05-11

    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.

  18. Lipopolysaccharides (LPS), up-regulate the IL-1-mRNA and down-regulate the glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS)-mRNAs in astroglial primary cultures.

    PubMed

    Letournel-Boulland, M L; Fages, C; Rolland, B; Tardy, M

    1994-01-01

    The effect of lipopolysaccharides (LPS), a component of gram-negative bacteria, has been studied in both exponentially growing and confluent morphologically differentiated astroglial cells in primary cultures. The expression of glial fibrillary acidic protein (GFAP) and Glutamine Synthetase (GS) were investigated in parallel with proliferation and expression of IL-1 beta-mRNA. During the exponential growth, proliferation was severely inhibited by LPS. The effect was time- and dose-dependent. On confluent differentiated cells LPS induced an inhibition of cell proliferation which was associated with a down-regulation of GFAP-mRNA, GS-mRNA and GS expressions and with a transitory increase in IL-1 beta mRNA expression. The observed effects might interact with the astroglial developmental program and with the astroglial function.

  19. Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation

    PubMed Central

    Euro, Liliya; Konovalova, Svetlana; Asin-Cayuela, Jorge; Tulinius, Már; Griffin, Helen; Horvath, Rita; Taylor, Robert W.; Chinnery, Patrick F.; Schara, Ulrike; Thorburn, David R.; Suomalainen, Anu; Chihade, Joseph; Tyynismaa, Henna

    2015-01-01

    The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes. PMID:25705216

  20. Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis.

    PubMed

    Niva, Cintia Carla; Lee, Jae Min; Myohara, Maroko

    2008-01-01

    Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4-5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61-71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion

  1. Beneficial consequences of a selective glutamine synthetase inhibitor in oats and legumes

    SciTech Connect

    Langston-Unkefer, P.J.; Knight, T.J.; Sengupta-Gopalan, C.

    1988-01-01

    We report on the effects of administering a unique glutamine synthetase inhibitor to cereals and N/sub 2/-fixing legumes. A bacterium (Pseudomonas syringae pv. tabaci) delivers this inhibitor to provide extended treatment periods; we inoculated the root systems of oat and legume plants with pv. tabaci to provide for delivery of this inhibitor to their root or root/nodule systems. Inoculation of legumes is accompanied by increased plant growth, total plant nitrogen, nodulation, and nitrogen fixation activity. Inoculation of the oats is accompanied by either of two results depending upon the genotype of the oat plant. One result is inhibition of plant growth followed by plant death as consequences of the loss of all of the glutamine synthetase activities in the plant and the subsequent accumulation of ammonia and cessation of nitrate uptake. The second and opposite result is observed in a small population of oats screened from a commercial cultivar and includes increased plant growth and leaf protein. The effects of this inhibitor can be beneficial when applied to appropriate plant material. In an attempt to effectively communicate these findings to the reader, we first introduce the inhibitor (a novel amino acid) and its bacterial delivery systems, the target of the inhibitor (glutamine synthetase-catalyzed ammonia assimilation), and the two different nitrogen economics in the legume and cereal plants used experimentally. The physiological, biochemical, and molecular genetic consequences of the inhibitor action in cereals and legumes, as we presently understand them, are then presented. 18 refs., 4 figs., 3 tabs.,

  2. Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

    PubMed

    Dar, Asif A; Pradhan, Trupti N; Kulkarni, Dakshayni P; Shah, Sagar U; Rao, Kanury V; Chaukar, Devendra A; D'Cruz, Anil K; Chiplunkar, Shubhada V

    2016-02-01

    Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions. PMID:26595239

  3. Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

    PubMed

    Dar, Asif A; Pradhan, Trupti N; Kulkarni, Dakshayni P; Shah, Sagar U; Rao, Kanury V; Chaukar, Devendra A; D'Cruz, Anil K; Chiplunkar, Shubhada V

    2016-02-01

    Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

  4. Biosynthetic engineering of nonribosomal peptide synthetases.

    PubMed

    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.

  5. Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P. falciparum.

    PubMed

    Khan, Sameena; Sharma, Arvind; Jamwal, Abhishek; Sharma, Vinay; Pole, Anil Kumar; Thakur, Kamal Kishor; Sharma, Amit

    2011-01-01

    Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation, and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.

  6. Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation.

    PubMed

    Bott, Alex J; Peng, I-Chen; Fan, Yongjun; Faubert, Brandon; Zhao, Lu; Li, Jinyu; Neidler, Sarah; Sun, Yu; Jaber, Nadia; Krokowski, Dawid; Lu, Wenyun; Pan, Ji-An; Powers, Scott; Rabinowitz, Joshua; Hatzoglou, Maria; Murphy, Daniel J; Jones, Russell; Wu, Song; Girnun, Geoffrey; Zong, Wei-Xing

    2015-12-01

    c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

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

    PubMed

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

    2014-12-01

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

  8. Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

    PubMed Central

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

    2014-01-01

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins. PMID:25536042

  9. Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.

    PubMed

    Yang, G; Sandalova, T; Lohman, K; Lindqvist, Y; Rendina, A R

    1997-04-22

    Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and <0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S

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

    NASA Astrophysics Data System (ADS)

    Kung, Irene Yuk Man

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

  11. Benadrostin, new inhibitor of poly(ADP-ribose) synthetase, produced by actinomycetes. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

    PubMed

    Aoyagi, T; Yoshida, S; Harada, S; Okuyama, A; Nakayama, C; Yoshida, T; Hamada, M; Takeuchi, T; Umezawa, H

    1988-08-01

    Benadrostin, a new inhibitor of poly(ADP-ribose) synthetase was discovered in the fermentation broth of Streptomyces flavovirens MH499-O'F1. It was purified by chromatography followed by solvent extraction and then isolated as colorless prisms. Benadrostin has the molecular formula of C8H5NO4. It was competitive with the substrate, and the inhibition constant (Ki) was 34 microM. PMID:3139601

  12. Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase

    SciTech Connect

    Hupe, D.J.; Behrens, N.D.; Boltz, R. )

    1990-09-01

    L-651,582, 5-amino-(4-(4-chlorobenzoyl)-3,5-dichlorobenzyl)-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.

  13. The evolution of Class II Aminoacyl-tRNA synthetases and the first code.

    PubMed

    Smith, Temple F; Hartman, Hyman

    2015-11-30

    Class II Aminoacyl-tRNA synthetases are a set of very ancient multi domain proteins. The evolution of the catalytic domain of Class II synthetases can be reconstructed from three peptidyl-hairpins. Further evolution from this primordial catalytic core leads to a split of the Class II synthetases into two divisions potentially associated with the operational code. The earliest form of this code likely coded predominantly Glycine (Gly), Proline (Pro), Alanine (Ala) and "Lysine"/Aspartic acid (Lys/Asp). There is a paradox in these synthetases beginning with a hairpin structure before the Genetic Code existed. A resolution is found in the suggestion that the primordial Aminoacyl synthetases formed in a transition from a Thioester world to a Phosphate ester world. PMID:26472323

  14. MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases.

    PubMed

    Felnagle, Elizabeth A; Barkei, John J; Park, Hyunjun; Podevels, Angela M; McMahon, Matthew D; Drott, Donald W; Thomas, Michael G

    2010-10-19

    The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.

  15. Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

    PubMed Central

    Köhrer, Caroline; Sullivan, Eric L.; RajBhandary, Uttam L.

    2004-01-01

    We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells. PMID:15576346

  16. Acid Rain: Activities for Science Teachers.

    ERIC Educational Resources Information Center

    Johnson, Eric; And Others

    1983-01-01

    Seven complete secondary/college level acid rain activities are provided. Activities include overview; background information and societal implications; major concepts; student objectives; vocabulary/material lists; procedures; instructional strategies; and questions/discussion and extension suggestions. Activities consider effects of acid rain on…

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

    SciTech Connect

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

    1990-05-01

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

  18. Glutamine synthetase in liver of the American alligator, Alligator mississippiensis.

    PubMed

    Smith, D D; Campbell, J W

    1987-01-01

    Glutamine synthetase was shown to be localized in liver mitochondria of the American alligator, Alligator mississippiensis, by immunofluorescent staining of frozen liver sections and by the detection of enzymatic activity and immunoreactive protein in the mitochondrial fraction following subcellular fractionation of liver tissue by differential centrifugation. The primary translation product of alligator liver glutamine synthetase mRNA was shown to have an Mr = 45,000 which is similar if not identical in size to that of the mature subunit. This mRNA was found to be heterogeneous in size with a major form corresponding to 2.8-3.0 kb and a lesser form corresponding to around 2 kb. Both are in excess of the size required to code for the glutamine synthetase subunit. The synthesis and presumably the mitochondrial import of glutamine synthetase in alligator liver are thus very similar to the same processes in avian liver. Despite the excretion of a high percentage of nitrogen as ammonia, the demonstration of a mitochondrial glutamine synthetase indicates the alligator has the typical avian-type uricotelic ammonia-detoxification system in liver. This suggests that the transition to uricotelism occurred in the sauropsid line of evolution and has persisted through both the lepidosaurian (snakes, lizards) and archosaurian (dinosaurs, crocodilians, birds) lines.

  19. Activation of carboxylic acids in asymmetric organocatalysis.

    PubMed

    Monaco, Mattia Riccardo; Poladura, Belén; Diaz de Los Bernardos, Miriam; Leutzsch, Markus; Goddard, Richard; List, Benjamin

    2014-07-01

    Organocatalysis, catalysis using small organic molecules, has recently evolved into a general approach for asymmetric synthesis, complementing both metal catalysis and biocatalysis. Its success relies to a large extent upon the introduction of novel and generic activation modes. Remarkably though, while carboxylic acids have been used as catalyst directing groups in supramolecular transition-metal catalysis, a general and well-defined activation mode for this useful and abundant substance class is still lacking. Herein we propose the heterodimeric association of carboxylic acids with chiral phosphoric acid catalysts as a new activation principle for organocatalysis. This self-assembly increases both the acidity of the phosphoric acid catalyst and the reactivity of the carboxylic acid. To illustrate this principle, we apply our concept in a general and highly enantioselective catalytic aziridine-opening reaction with carboxylic acids as nucleophiles.

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

    PubMed

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

    2016-07-01

    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.

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

    PubMed Central

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

    2016-01-01

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

  2. Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

    PubMed

    Savilov, P N; Yakovlev, V N

    2016-01-01

    Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

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

    DOEpatents

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

    2006-08-01

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

  4. Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    DOEpatents

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

    2012-05-08

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

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

    SciTech Connect

    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.

  6. Determinants within the C-terminal domain of Streptomyces lividans acetyl-CoA synthetase that block acetylation of its active site lysine in vitro by the protein acetyltransferase (Pat) enzyme.

    PubMed

    Tucker, Alex C; Escalante-Semerena, Jorge C

    2014-01-01

    Reversible lysine acetylation (RLA) is a widespread regulatory mechanism that modulates the function of proteins involved in diverse cellular processes. A strong case has been made for RLA control exerted by homologues of the Salmonella enterica protein acetyltransferase (SePat) enzyme on the broadly distributed AMP-forming CoA ligase (a.k.a. acyl-CoA synthetases) family of metabolic enzymes, with acetyl-CoA synthetase (Acs) being the paradigm in the field. Here we investigate why the Acs homologue in Streptomyces lividans (SlAcs) is poorly acetylated in vitro by the S. lividans protein acetyltransferase (SlPat) enzyme. Chimeras of S. enterica Acs (SeAcs) and S. lividans Acs (SlAcs) constructed during the course of this work were acetylated by SlPatA in vitro, retained most of their activity, and were under RLA control in a heterologous host. We identified SeAcs residues N- and C-terminal to the target lysine that when introduced into SlAcs, rendered the latter under RLA control. These results lend further support to the idea that Pat enzymes interact with extensive surfaces of their substrates. Finally, we suggest that acetylation of SlAcs depends on factors or conditions other than those present in our in vitro system. We also discuss possible explanations why SlAcs is not controlled by RLA as defined in other bacterial species.

  7. The McbB component of microcin B17 synthetase is a zinc metalloprotein.

    PubMed

    Zamble, D B; McClure, C P; Penner-Hahn, J E; Walsh, C T

    2000-12-26

    The microcin B17 synthetase converts glycine, serine, and cysteine residues in a polypeptide precursor into oxazoles and thiazoles during the maturation of the Escherichia coli antibiotic Microcin B17. This multimeric enzyme is composed of three subunits (McbB, McbC, and McbD), and it employs both ATP and FMN as cofactors. The McbB subunit was purified as a fusion with the maltose-binding protein (MBP), and metal analysis revealed that this protein binds 0.91+/-0.17 zinc atoms. Upon incubation of MBP-McbB with excess zinc, the stoichiometry increased to two atoms of zinc bound, but metal binding to the second site resulted in a decrease in the heterocyclization activity when MBP-McbB was reconstituted with the other components of the synthetase. Apo-protein was prepared by using p-hydroxymercuriphenylsulfonic acid (PMPS), and loss of the metal caused a severe reduction in enzymatic activity. However, if dithiothreitol was added to the PMPS reactions within a few minutes, enzymatic activity was retained and MBP-McbB could be reconstituted with zinc. Spectroscopic analysis of the cobalt-containing protein and extended X-ray absorption fine structure analysis of the zinc-containing protein both provide evidence for a tetrathiolate coordination sphere. Site-directed mutants of MBP-McbB as well as the synthetase tagged with the calmodulin-binding peptide were constructed. Activity assays and metal analysis were used to determine which of the six cysteines in McbB are metal ligands. These results suggest that the zinc cofactor in McbB plays a structural role.

  8. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria.

    PubMed

    Yao, Jiangwei; Bruhn, David F; Frank, Matthew W; Lee, Richard E; Rock, Charles O

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

  9. Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells

    PubMed Central

    Shan, Jixiu; Hamazaki, Takashi; Tang, Tiffany A.; Terada, Naohiro

    2013-01-01

    In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages. PMID:23736538

  10. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  11. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.

    PubMed

    Verlander, Jill W; Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E; Weiner, I David

    2013-09-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.

  12. L-arginine recognition by yeast arginyl-tRNA synthetase.

    PubMed Central

    Cavarelli, J; Delagoutte, B; Eriani, G; Gangloff, J; Moras, D

    1998-01-01

    The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs. PMID:9736621

  13. Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

    PubMed

    Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A G; Santamaría-Gómez, Javier; Patterson, Carl J; Foster, Andrew W; Bru-Martínez, Roque; Robinson, Nigel J; Luque, Ignacio

    2015-11-16

    Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

  14. Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress

    PubMed Central

    Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A. G.; Santamaría-Gómez, Javier; Patterson, Carl J.; Foster, Andrew W.; Bru-Martínez, Roque; Robinson, Nigel J.; Luque, Ignacio

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNAThr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNAThr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs. PMID:26464444

  15. Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

    PubMed

    Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A G; Santamaría-Gómez, Javier; Patterson, Carl J; Foster, Andrew W; Bru-Martínez, Roque; Robinson, Nigel J; Luque, Ignacio

    2015-11-16

    Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs. PMID:26464444

  16. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  17. Building biologically active nucleic acid nanocomplexes.

    PubMed

    Smith, C I Edvard; Lundin, Karin E; Simonson, Oscar E; Moreno, Pedro M D; Svahn, Mathias G; Wenska, Malgorzata; Strömberg, Roger

    2008-01-01

    The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.

  18. Biosynthetic engineering of nonribosomal peptide synthetases.

    PubMed

    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. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase.

    PubMed

    Wang, Dezheng; Wang, Cheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-01-01

    Glutathione (GSH) is an important bioactive substance applied widely in pharmaceutical and food industries. Due to the strong product inhibition in the GSH biosynthetic pathway, high levels of intracellular content, yield and productivity of GSH are difficult to achieve. Recently, a novel bifunctional GSH synthetase was identified to be less sensitive to GSH. A recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus was constructed for GSH production. In this study, efficient GSH production using this engineered strain was investigated. The cultivation process was optimized by controlling dissolved oxygen (DO), amino acid addition and glucose feeding. 36.8 mM (11.3 g/L) GSH were formed at a productivity of 2.06 mM/h when the amino acid precursors (75 mM each) were added and glucose was supplied as the sole carbon and energy source. PMID:26586402

  20. Fatty acids regulate CREBh via transcriptional mechanisms that are dependent on proteasome activity and insulin

    PubMed Central

    Gentile, CL; Wang, D; Pfaffenbach, KT; Cox, R; Wei, Y; Pagliassotti, MJ

    2011-01-01

    Excess fatty acids are closely associated with metabolic dysfunction. The deleterious effects of fatty acids relate, in part, to their ability to up-regulate proinflammatory cytokines and propagate a state of systemic inflammation. CREBh is a recently identified transcription factor that appears to be required for hepatic synthesis of C-reactive protein (CRP). Recent data suggest that fatty acids can up-regulate CREBh, thus establishing a potential molecular link between fatty acids and inflammation. The aim of the current study was to examine the nature and mechanisms of fatty acid-mediated regulation of CREBh. H4IIE liver cells were incubated in the absence or presence of varying concentrations (50–500 μM) of albumin-bound, long-chain saturated (palmitate, stearate) or unsaturated (oleate, linoleate) fatty acids (1–16 hours). All fatty acids significantly increased CREBh gene expression via transcriptional mechanisms, at concentrations as low as 50 μM. Palmitate- or oleate-mediated upregulation of CREBh was not inhibited by triacsin C, an inhibitor of long-chain fatty acyl CoA synthetase, or by the PPARα antagonist, MK886. Inhibition of proteasome activity with MG132 or lactacystin, or inclusion of insulin reduced palmitate- and oleate-mediated increases in CREBh mRNA. Finally, we examined fatty acid regulation of CREBh in vivo. Male Wistar rats were exposed to a 4-hour pancreatic clamp combined with infusion of saline or a mixed lipid emulsion. CREBh mRNA and protein were significantly increased in rats exposed to the lipid infusion compared to the saline group. Collectively, these results may have important implications for metabolic diseases characterized by excess fatty acids, insulin resistance and inflammation. PMID:20607591

  1. Zymographic detection of cinnamic acid decarboxylase activity.

    PubMed

    Prim, Núria; Pastor, F I Javier; Diaz, Pilar

    2002-11-01

    The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

  2. Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

    PubMed Central

    Krauss, S. W.; Stollar, B. D.; Friedkin, M.

    1973-01-01

    Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene. Images PMID:4575286

  3. Cocrystal Structures of Glycyl-tRNA Synthetase in Complex with tRNA Suggest Multiple Conformational States in Glycylation*

    PubMed Central

    Qin, Xiangjing; Hao, Zhitai; Tian, Qingnan; Zhang, Zhemin; Zhou, Chun; Xie, Wei

    2014-01-01

    Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease. PMID:24898252

  4. Cocrystal structures of glycyl-tRNA synthetase in complex with tRNA suggest multiple conformational states in glycylation.

    PubMed

    Qin, Xiangjing; Hao, Zhitai; Tian, Qingnan; Zhang, Zhemin; Zhou, Chun; Xie, Wei

    2014-07-18

    Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease.

  5. Dietary lipid levels impact lipoprotein lipase, hormone-sensitive lipase, and fatty acid synthetase gene expression in three tissues of adult GIFT strain of Nile tilapia, Oreochromis niloticus.

    PubMed

    Tian, Juan; Wu, Fan; Yang, Chang-Geng; Jiang, Ming; Liu, Wei; Wen, Hua

    2015-02-01

    The objective of this study was to assess the effects of dietary lipids on growth performance, body composition, serum parameters, and expression of genes involved in lipid metabolism in adult genetically improved farmed tilapia (GIFT strain) of Nile tilapia, Oreochromis niloticus. We randomly assigned adult male Nile tilapia (average initial body weight = 220.00 ± 9.54 g) into six groups consisting of four replicates (20 fish per replicate). Fish in each group were hand-fed a semi-purified diets containing different lipid levels [3.3 (the control group), 28.4, 51.4, 75.4, 101.9, and 124.1 g kg(-1)] for 8 weeks. The results indicated that there was no obvious effect in feeding rate among all groups (P > 0.05). The highest weight gain, specific growth rate, and protein efficiency ratio in 75.4 g kg(-1) diet group were increased by 23.31, 16.17, and 22.02 % than that of fish in the control group (P < 0.05). Protein retention ratio was highest in 51.4 g kg(-1) diet group. The results revealed that the optimum dietary lipid level for maximum growth performance is 76.6-87.9 g kg(-1). Increasing dietary lipid levels contributed to increased tissue and whole body lipid levels. Saturated and monounsaturated fatty acids (MUFAs) decreased, and polyunsaturated fatty acids increased with increasing dietary lipid levels. With the exception of MUFAs, the fatty acid profiles of liver and muscle were similar. Dietary lipid levels were negatively correlated with low-density lipoprotein- cholesterol content and positively with triacylglycerol and glucose contents. In the lipid-fed groups, there was a significant down-regulation of fatty acid synthase (FAS) mRNA in liver, muscle, and visceral adipose tissues. There was a rapid up-regulation of lipoprotein lipase (LPL) mRNA in muscle and liver with increasing dietary lipid levels. In visceral adipose tissue, LPL mRNA was significantly down-regulated in the lipid-fed groups. Dietary lipids increased hormone-sensitive lipase (HSL) m

  6. Bacterial and eukaryotic phenylalanyl-tRNA synthetases catalyze misaminoacylation of tRNA(Phe) with 3,4-dihydroxy-L-phenylalanine.

    PubMed

    Moor, Nina; Klipcan, Liron; Safro, Mark G

    2011-10-28

    Aminoacyl-tRNA synthetases exert control over the accuracy of translation by selective pairing the correct amino acids with their cognate tRNAs, and proofreading the misacylated products. Here we show that three existing, structurally different phenylalanyl-tRNA synthetases-human mitochondrial (HsmtPheRS), human cytoplasmic (HsctPheRS), and eubacterial from Thermus thermophilus (TtPheRS), catalyze mischarging of tRNA(Phe) with an oxidized analog of tyrosine-L-dopa. The lowest level of L-dopa discrimination over the cognate amino acid, exhibited by HsmtPheRS, is comparable to that of tyrosyl-tRNA synthetase. HsmtPheRS and TtPheRS complexes with L-dopa revealed in the active sites an electron density shaping this ligand. HsctPheRS and TtPheRS possessing editing activity are capable of hydrolyzing the exogenous L-dopa-tRNA(Phe) as efficiently as Tyr-tRNA(Phe). However, editing activity of PheRS does not guarantee reduction of the aminoacylation error rate to escape misincorporation of L-dopa into polypeptide chains.

  7. Effect of thromboxane synthetase inhibitor on feline infectious peritonitis in cats.

    PubMed

    Watari, T; Kaneshima, T; Tsujimoto, H; Ono, K; Hasegawa, A

    1998-05-01

    Two cats with abdominal effusion and anorexia were diagnosed as feline infectious peritonitis (FIP). We tried to evaluate the effect of thromboxane (Tx) synthetase inhibitor, ozagrel hydrochloride, on the progression of symptoms and clinicopathologic data characteristic to FIP. After administration of Tx synthetase inhibitor, improvement of appetite and activity, decreases of peritoneal effusion, reduction of leukocyte number to normal level, and improvement of hyper gamma-globulinemia were found in 2 cats with FIP. These findings suggest that the vasculitis in FIP can be successfully treated with Tx synthetase inhibitor which inhibits platelet aggregation.

  8. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  9. CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila

    PubMed Central

    Ermanoska, Biljana; Motley, William W.; Leitão-Gonçalves, Ricardo; Asselbergh, Bob; Lee, LaTasha H.; De Rijk, Peter; Sleegers, Kristel; Ooms, Tinne; Godenschwege, Tanja A.; Timmerman, Vincent; Fischbeck, Kenneth H.; Jordanova, Albena

    2014-01-01

    Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth disease (CMT). The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 – genes identified in a retinal degeneration screen to modify mutant YARS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies. PMID:24807208

  10. A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate.

    PubMed

    Ratner, S

    1983-12-01

    A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions. PMID:6660522

  11. Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

    PubMed Central

    Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

    2014-01-01

    Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

  12. The Enterococcal Cytolysin Synthetase Coevolves with Substrate for Stereoselective Lanthionine Synthesis.

    PubMed

    Tang, Weixin; Thibodeaux, Gabrielle N; van der Donk, Wilfred A

    2016-09-16

    Stereochemical control is critical in natural product biosynthesis. For ribosomally synthesized and post-translationally modified peptides (RiPPs), the mechanism(s) by which stereoselectivity is achieved is still poorly understood. In this work, we focused on the stereoselective lanthionine synthesis in lanthipeptides, a major class of RiPPs formed by the addition of Cys residues to dehydroalanine (Dha) or dehydrobutyrine (Dhb). Nonenzymatic cyclization of the small subunit of a virulence lanthipeptide, the enterococcal cytolysin, resulted in the native modified peptide as the major product, suggesting that both regioselectivity and stereoselectivity are inherent to the dehydrated peptide sequence. These results support previous computational studies that a Dhx-Dhx-Xxx-Xxx-Cys motif (Dhx = Dha or Dhb; Xxx = any amino acid except Dha, Dhb, and Cys) preferentially cyclizes by attack on the Re face of Dha or Dhb. Characterization of the stereochemistry of the products formed enzymatically with substrate mutants revealed that the lanthionine synthetase actively reinforces Re face attack. These findings support the hypothesis of substrate-controlled selectivity in lanthionine synthesis but also reveal likely coevolution of substrates and lanthionine synthetases to ensure the stereoselective synthesis of lanthipeptides with defined biological activities. PMID:27348535

  13. β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis

    PubMed Central

    Gaudelli, Nicole M.; Long, Darcie H.; Townsend, Craig A.

    2014-01-01

    Non-ribosomal peptide synthetases (NRPSs) are giant enzymes comprised of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks.1 The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and nocardicins. Although penicillins and cephalosporins are synthesised from a classically derived NRPS tripeptide (from ACVS, δ-(L-α-aminoadipyl)–L-cysteinyl–D-valine synthetase)2, we now report an unprecedented NRPS activity to both assemble a serine-containing peptide and mediate its cyclisation to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation (C) domain, which typically carries out peptide bond formation during product assembly, was found to also synthesise the embedded 4-membered ring. Here, a mechanism is proposed and supporting experiments are described, which is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.3,4 PMID:25624104

  14. Molecular definition of bovine argininosuccinate synthetase deficiency.

    PubMed Central

    Dennis, J A; Healy, P J; Beaudet, A L; O'Brien, W E

    1989-01-01

    Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia. Images PMID:2813370

  15. Aminoacyl-tRNA Synthetase Complexes in Evolution

    PubMed Central

    Havrylenko, Svitlana; Mirande, Marc

    2015-01-01

    Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis. PMID:25807264

  16. The aminoacyl-tRNA synthetases of Drosophila melanogaster.

    PubMed

    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

  17. The aminoacyl-tRNA synthetases of Drosophila melanogaster

    PubMed Central

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

    2015-01-01

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

  18. Function of the major synthetase subdomains of carbamyl-phosphate synthetase.

    PubMed

    Guy, H I; Evans, D R

    1996-06-01

    The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex. PMID:8662713

  19. Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B.

    PubMed

    Boehlein, S K; Walworth, E S; Richards, N G; Schuster, S M

    1997-05-01

    Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

  20. Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure.

    PubMed

    Chew, Shit F; Ong, Tan F; Ho, Lilian; Tam, Wai L; Loong, Ai M; Hiong, Kum C; Wong, Wai P; Ip, Yuen K

    2003-10-01

    Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air

  1. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.

    PubMed

    Wan, Wei; Tharp, Jeffery M; Liu, Wenshe R

    2014-06-01

    The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA(Pyl). Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA(Pyl). These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs. PMID:24631543

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

    SciTech Connect

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

    1982-04-01

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

  3. Aminoacyl-tRNA Synthetases in the Bacterial World.

    PubMed

    Giegé, Richard; Springer, Mathias

    2016-05-01

    Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation. Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g., in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show huge structural plasticity related to function and limited idiosyncrasies that are kingdom or even species specific (e.g., the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS). Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably between distant groups such as Gram-positive and Gram-negative Bacteria. The review focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation, and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulated in last two decades is reviewed, showing how the field moved from essentially reductionist biology towards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRS paralogs (e.g., during cell wall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointed throughout the review and

  4. Energetics of S-adenosylmethionine synthetase catalysis.

    PubMed

    McQueney, M S; Anderson, K S; Markham, G D

    2000-04-18

    S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent. The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PP(i)), and phosphate (P(i)) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, and equilibrium binding measurements in conjunction with computer modeling. These studies provided the rate constants for substrate binding, the two chemical interconversion steps [AdoMet formation and subsequent tripolyphosphate (PPP(i)) hydrolysis], and product release. The data demonstrate the presence of a kinetically significant isomerization of the E.AdoMet.PP(i).P(i) complex before product release. The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies. In contrast, the subsequent PPP(i) hydrolytic reaction is energetically better balanced. The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPP(i) hydrolysis to allow enzyme turnover. Crystallographic studies have shown that a mobile protein loop gates access to the active site. The present kinetic studies indicate that this loop movement is rapid with respect to k(cat) and with respect to substrate binding at physiological concentrations. The uniformly slow binding rates of 10(4)-10(5) M(-)(1) s(-)(1) for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced. PMID:10757994

  5. NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharides in Group B Streptococcus

    PubMed Central

    Lewis, Amanda L.; Cao, Hongzhi; Patel, Silpa K.; Diaz, Sandra; Ryan, Wesley; Carlin, Aaron F.; Thon, Vireak; Lewis, Warren G.; Varki, Ajit; Chen, Xi; Nizet, Victor

    2008-01-01

    Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide (CPS). Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) was enhanced by CTP and Mg2+, the substrate and co-factor respectively of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bi-functional NeuA esterase from E. coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac2, followed by CMP-activation of Neu5Ac; or, activation of Neu5,9Ac2, then de-O-acetylation of CMP-Neu5,9Ac2. Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and over-expression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity, but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of CPS Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria, and provide a genetic strategy for manipulating GBS O-acetylation, in order to explore the role of this modification in GBS pathogenesis and immunogenicity. PMID:17646166

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

    PubMed Central

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

    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 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

  7. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    SciTech Connect

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  8. CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

    PubMed Central

    Chang, Yu-Fang; Carman, George M.

    2008-01-01

    CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C. PMID:18439916

  9. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

    PubMed

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M R K; Freund, Yvonne R; DeRisi, Joseph; Cusack, Stephen; Rosenthal, Philip J

    2016-08-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

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

    SciTech Connect

    Bonnefond, Luc; Frugier, Magali; Touzé, Elodie; Lorber, Bernard; Florentz, Catherine; Giegé, Richard Rudinger-Thirion, Joëlle; Sauter, Claude

    2007-04-01

    Crystals of human mitochondrial tyrosyl-tRNA synthetase lacking the C-terminal S4-like domain diffract to 2.7 Å resolution and are suitable for structure determination. Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA{sup Tyr} charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4{sub 3}2{sub 1}2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 Å resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

  11. Neurospora crassa mutants deficient in asparagine synthetase.

    PubMed Central

    MacPhee, K G; Nelson, R E; Schuster, S M

    1983-01-01

    Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase. PMID:6137480

  12. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

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

    PubMed

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

    2015-02-01

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

  14. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  15. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population. PMID:23656379

  16. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.

  17. Regulation of the stability of poly(I)xpoly(C)-induced human fibroblast interferon mRNA: selective inactivation of interferon mRNA and lack of involvement of 2',5'-oligo(A) synthetase activation during the shutoff of interferon production.

    PubMed

    Sehgal, P B; Gupta, S L

    1980-06-01

    The inactivation of interferon mRNA during the shutoff phase of interferon production in poly(I)xpoly(C)-induced human fibroblast cultures is selective. We have determined that the shutoff of interferon production, which takes place from 3 to 8 hr after the beginning of induction, is not associated with an appreciable declined in the rate of bulk cellular protein synthesis or of cellular protein secretion. While the amount of translatable interferon mRNA declined markedly during the shutoff phase, the level of translatable bulk cellular mRNA and the stability of [3H]uridine-labeled mRNA were unaffected. Superinduction with actinomycin D selectively stabilized interferon mRNA with no apparent effect on the stability of bulk cellular mRNA. Furthermore, an activation of the 2',5'-oligo(A) synthetase/endonuclease system does not appear to be involved in the shutoff phenomenon. Uninduced FS-4 cells contained a low basal level of 2'5'-oligo(A) synthetase activity, which was unchanged in poly(I)xpoly(C)-induced cells during the shutoff phase. Treatment of FS-4 cells with interferon for 16-18 hr prior to induction increased the enzyme activity by approximately 200-fold. However, this did not inhibit interferon production after induction with poly(I)xpoly(C) alone or after superinduction with cycloheximide or actinomycin D or both. Furthermore, the rates of decay of interferon production were comparable in cells with either a basal or an increased level of 2',5'-oligo(A) synthetase. Thus a 200-fold increase in 2',5'-oligo(A) synthetase level did not affect either the stability of interferon mRNA or the efficacy of interferon superinduction by metabolic inhibitors.

  18. Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase

    PubMed Central

    Fang, Pengfei; Yu, Xue; Jeong, Seung Jae; Mirando, Adam; Chen, Kaige; Chen, Xin; Kim, Sunghoon; Francklyn, Christopher S.; Guo, Min

    2015-01-01

    The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth ‘orthogonal’ subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes. PMID:25824639

  19. Elevated hepatic gamma-glutamylcysteine synthetase activity and abnormal sulfate levels in liver and muscle tissue may explain abnormal cysteine and glutathione levels in SIV-infected rhesus macaques.

    PubMed

    Gross, A; Hack, V; Stahl-Hennig, C; Dröge, W

    1996-11-20

    To establish whether the low cysteine and glutathione levels in HIV-infected patients and SIV-infected rhesus macaques may be consequences of an abnormal cysteine catabolism, we analyzed sulfate and glutathione levels in macaques. Muscle tissue (m. vastus lateralis and m. gastrocnemius) of SIV-infected macaques (n = 25) had higher sulfate and lower glutathione and glutamate levels than that of uninfected controls (n =9). Hepatic tissue, in contrast, showed decreased sulfate and glutathione disulfide (GSSG) levels, and increased gamma-glutamylcysteine synthetase (gamma-GCS) activity. These findings suggest drainage of the cysteine pool by increased cysteine catabolism in skeletal muscle tissue, and by increased hepatic glutathione biosynthesis. Cachectic macaques also showed increased urea levels and decreased glutamine/urea ratios in the liver, which are obviously related to the abnormal urea excretion and negative nitrogen balance commonly observed in cachexia. As urea production and net glutamine synthesis in the liver are strongly influenced by proton-generating processes, the abnormal hepatic urea production may be the direct consequence of the cysteine deficiency and the decreased catabolic conversion of cysteine into sulfate and protons in the liver.

  20. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

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

    PubMed

    Murdeshwar, Maya S; Chatterji, Dipankar

    2012-08-01

    In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

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

    PubMed Central

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

    2000-01-01

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

  3. Photoinduced biochemical activity of fullerene carboxylic acid

    SciTech Connect

    Tokuyama, Hidetoshi; Yamago, Shigeru; Nakamura, Eiichi; Shiraki, Takashi; Sugiura, Yukio

    1993-08-25

    Here we report the preparation of a water-miscible fullerene carboxylic acid (2) and its biological activity-cytotoxicity and G-selective DNA cleaving ability. What is truly remarkable is that the biological activity of C{sub 60} was observed only under irradiation with visible light and not in the dark, suggesting that fullerenes may serve as useful photosensitive biochemical probes. We have found, for the first time, that even low-energy visible light is surfficient to induce biological activity in fullerene derivatives. Among the numerous implications of the present findings, the most exciting prospect includes the use of fullerene derivatives for photodynamic therapy. 18 refs., 2 figs., 1 tab.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

    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. Identification and molecular characterization of acyl-CoA synthetase in human erythrocytes and erythroid precursors.

    PubMed

    Malhotra, K T; Malhotra, K; Lubin, B H; Kuypers, F A

    1999-11-15

    Full-length cDNA species encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukaemic cell line were cloned, sequenced and expressed. The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase with high similarity to rat brain ACS2 (91% identical). The second form (66% identical with LACS5) was 97% identical with human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kb mRNA transcript in human haemopoietic stem cells from cord blood, bone marrow, reticulocytes and fetal blood cells derived from fetal liver. An additional 6.3 kb transcript is also found in these erythrocyte precursors; 2.9 and 9.6 kb transcripts of LACS5 are found in human brain, but transcripts are virtually absent from human heart, kidney, liver, lung, pancreas, spleen and skeletal muscle. The 78 kDa expressed LACS5 protein used the long-chain fatty acids palmitic acid, oleic acid and arachidonic acid as substrates. Antibodies directed against LACS5 cross-reacted with erythrocyte membranes. We conclude that early erythrocyte precursors express at least two different forms of acyl-CoA synthetase and that LACS5 is present in mature erythrocyte plasma membranes.

  7. Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase.

    PubMed Central

    Fraisl, Peter; Forss-Petter, Sonja; Zigman, Mihaela; Berger, Johannes

    2004-01-01

    It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy). The precise function of the ALDP (ALD protein) still remains unclear. Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids). As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene. In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals. During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation. Protein fractionation and confocal laser imaging of Bgm-green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi. Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment. Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference. Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely. PMID:14516277

  8. Nineteen-year follow-up of a patient with severe glutathione synthetase deficiency.

    PubMed

    Atwal, Paldeep S; Medina, Casey R; Burrage, Lindsay C; Sutton, V Reid

    2016-07-01

    Glutathione synthetase deficiency is a rare autosomal recessive disorder resulting in low levels of glutathione and an increased susceptibility to oxidative stress. Patients with glutathione synthetase deficiency typically present in the neonatal period with hemolytic anemia, metabolic acidosis and neurological impairment. Lifelong treatment with antioxidants has been recommended in an attempt to prevent morbidity and mortality associated with the disorder. Here, we present a 19-year-old female who was diagnosed with glutathione synthetase deficiency shortly after birth and who has been closely followed in our metabolic clinic. Despite an initial severe presentation, she has had normal intellectual development and few complications of her disorder with a treatment regimen that includes polycitra (citric acid, potassium citrate and sodium citrate), vitamin C, vitamin E and selenium.

  9. 19-Year Follow-up of A Patient With Severe Glutathione Synthetase Deficiency

    PubMed Central

    Atwal, Paldeep S.; Medina, Casey R.; Burrage, Lindsay C.; Sutton, V. Reid

    2016-01-01

    Glutathione synthetase deficiency is a rare autosomal recessive disorder resulting in low levels of glutathione and an increased susceptibility to oxidative stress. Patients with glutathione synthetase deficiency typically present in the neonatal period with hemolytic anemia, metabolic acidosis and neurological impairment. Lifelong treatment with antioxidants has been recommended in an attempt to prevent morbidity and mortality associated with the disorder. Here we present a 19-year-old female who was diagnosed with glutathione synthetase deficiency shortly after birth and who has been closely followed in our metabolic clinic. Despite an initial severe presentation, she has had normal intellectual development and few complications of her disorder with a treatment regimen that includes polycitra (citric acid, potassium citrate and sodium citrate), vitamin C, vitamin E and selenium. PMID:26984560

  10. Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase.

    PubMed

    Liu, X; Guy, H I; Evans, D R

    1994-11-01

    Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain

  11. Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes.

    PubMed Central

    Rosier, F; Lambert, D; Mertens-Strijthagen, M

    1996-01-01

    Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague-Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose-deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model. PMID:8615836

  12. Preparation of the multienzyme system gramicidin S-synthetase 2 with an aqueous three-phase system.

    PubMed

    Kirchner, A; Simonis, M; von Döhren, H

    1987-06-19

    The distribution of gramicidin S-synthetase activity from disrupted cells suspended in aqueous two- and three-phase systems was investigated. An optimized three-phase system containing 5% dextran, 8% Ficoll, 11% PEG and 6.7% disrupted cells was found to be effective in extracting gramicidin S-synthetase activity. The activity yield achieved was higher in comparison to other preparation methods, and the subsequent purification steps were greatly facilitated. The time needed for the preparation of the labile gramicidin S-synthetase was considerably reduced. The combination of the aqueous phase extraction with chromatographic methods yielded 19 mg gramicidin S-synthetase 2 in essentially pure form from 30 g (wet weight) of cells.

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

    SciTech Connect

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

    1987-05-01

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

  14. Expression of glutathione and gamma-glutamylcysteine synthetase mRNA is Jun dependent.

    PubMed

    Sekhar, K R; Meredith, M J; Kerr, L D; Soltaninassab, S R; Spitz, D R; Xu, Z Q; Freeman, M L

    1997-05-29

    The gene GLCLC encodes the catalytic subunit of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase E.C. 6.3.2.2), the rate limiting enzyme for glutathione synthesis. When HepG2 cells were exposed to the serine/threonine phosphatase inhibitor okadaic acid (OA), increased expression of GLCLC was observed, as was the development of resistance to xenobiotic induced GSH depletion. Okadaic acid is known to activate both NF-kappaB and AP-1 activity. Inhibition of NF-kappaB activity by overexpression of an IkappaB alpha transdominant inhibitor or exposure to the protease inhibitor TLCK did not inhibit the OA mediated increase in GLCLC transcripts. Fibroblasts derived from a mouse containing a c-Jun null mutation exhibited diminished AP-1 binding activity, reduced levels of GLCLC message, and a correspondingly low GSH concentration compared to wild type cells. When the null cells, which express Jun B and Jun D, were exposed to OA, AP-1 binding activity increased, as did expression of GLCLC message. These results indicate that AP-1 transcription factors participate in the regulation of glutathione metabolism.

  15. Further characterization of Escherichia coli alanyl-tRNA synthetase.

    PubMed

    Sood, S M; Slattery, C W; Filley, S J; Wu, M X; Hill, K A

    1996-04-15

    Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1. From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS). Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1. In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C. PMID:8645007

  16. Allosteric dominance in carbamoyl phosphate synthetase.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1999-02-01

    A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed. PMID:9931004

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

    PubMed

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

    2011-10-01

    The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state. PMID:21981925

  18. Inhibition of Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

    PubMed Central

    Merali, S; Zhang, Y; Sloan, D; Meshnick, S

    1990-01-01

    A new reversed-phase high-pressure liquid chromatography assay procedure for dihydropteroate synthetase (DHPS) that involves the elution of the enzyme incubation solution with a series of three solvents of decreasing polarity (ammonium phosphate buffer, 10% methanol, and 50% methanol) was designed. By this procedure DHPS was detected in Escherichia coli and Pneumocystis carinii with specific activities of 450 and 14 U/mg, respectively. A comparison of the effects of five sulfa drugs on P. carinii DHPS activity revealed that dapsone is the most potent of these drugs. PMID:2203302

  19. Isolation and characterization of rat liver microsomal R-ibuprofenoyl-CoA synthetase.

    PubMed

    Brugger, R; García Alía, B; Reichel, C; Waibel, R; Menzel, S; Brune, K; Geisslinger, G

    1996-10-11

    Microsomal long-chain acyl-CoA synthetase (EC 6.1.2.3.) has been suggested to be involved in the stereoselective formation of the CoA thioester of ibuprofen. In this study, we demonstrated that the microsomal enzyme from rat liver responsible for palmitoyl-CoA synthesis also catalyzes the formation of R-ibuprofenoyl-CoA in a Mg(2+)- and ATP-dependent process. Long-chain acyl-CoA synthetase from rat liver microsomes was purified to homogeneity as evidenced by SDS-gel electrophoresis. Simultaneous measurements of palmitoyl-CoA and R-ibuprofenoyl-CoA formation with HPLC in various fractions and purification steps during protein isolation revealed a high correlation between both activities. The purification procedure included solubilization of the microsomes obtained from rat livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, hydroxyapatite, and phosphocellulose. The purified enzyme exhibited an apparent molecular weight of 72 kDa as estimated by SDS gel electrophoresis, with specific activities of 71 nmol.min-1.mg-1 protein and 901 nmol.min-1.mg-1 protein for formation of R-ibuprofenoyl-CoA and palmitoyl-CoA, respectively. Palmitoyl-CoA formation catalyzed by the purified enzyme exhibited biphasic kinetics indicative of two isoforms, a high-affinity (KM 0.13 +/- 0.11 microM), low-capacity form and a low-affinity (KM 81 +/- 11.5 microM), high-capacity form. In contrast, measurement of R-ibuprofenoyl-CoA synthesis over a concentration range from 5 to 3000 microM showed the participation of a single CoA ligase with a KM of 184 +/- 19 microM, corresponding to the low-affinity isoform of palmitoyl-CoA synthesis with a marked enantioselectivity towards the R-form of ibuprofen. R-ibuprofenoyl-CoA formation of the enzyme preparation was inhibited by palmitic acid (KI 13.5 +/- 0.5 microM) and S-ibuprofen (KI 405 +/- 10 microM). In summary, these data give strong evidence for the identity of R-ibuprofenoyl-CoA and long

  20. Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

    PubMed

    Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

    2014-03-01

    Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.

  1. Activation of Nrf2-mediated oxidative stress response in macrophages by hypochlorous acid

    SciTech Connect

    Pi Jingbo Zhang Qiang; Woods, Courtney G.; Wong, Victoria; Collins, Sheila; Andersen, Melvin E.

    2008-02-01

    Hypochlorous acid (HOCl), a potent oxidant generated when chlorine gas reacts with water, is important in the pathogenesis of many disorders. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative damage. In the present study, the effect of HOCl on Nrf2 activation was investigated in macrophages, one of the target cells of chlorine gas exposure. Exposure of RAW 264.7 macrophages to HOCl resulted in increased protein levels of Nrf2 in nuclear extractions, as well as a time- and dose-dependent increase in the expression of Nrf2 target genes, including heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 (NQO-1), glutamate cysteine ligase catalytic subunit (GCLC), and glutathione synthetase (GS). Additionally, intracellular glutathione (GSH), which is the prime scavenger for HOCl in cells, decreased within the first hour of HOCl exposure. The decline was followed by a GSH rebound that surpassed the initial basal levels by up to 4-fold. This reversal in GSH levels closely correlated with the gene expression profile of GCLC and GS. To study the mechanisms of Nrf2 activation in response to HOCl exposure, we examined the effects of several antioxidants on Nrf2-mediated response. Pretreatment with cell-permeable catalase, N-acetyl-L-cysteine or GSH-monoethyl ester markedly reduced expression of NQO-1 and GCLC under HOCl challenge conditions, suggesting intracellular ROS-scavenging capacity affects HOCl-induced Nrf2 activation. Importantly, pre-activation of Nrf2 with low concentrations of pro-oxidants protected the cells against HOCl-induced cell damage. Taken together, we provide direct evidence that HOCl activates Nrf2-mediated antioxidant response, which protects cells from oxidative damage.

  2. Physiological and Kinetic Studies with Anthranilate Synthetase of Bacillus alvei1

    PubMed Central

    Catena, Anthony; DeMoss, R. D.

    1970-01-01

    Anthranilate synthetase from Bacillus alvei was partially purified by ammonium sulfate fractionation and was stabilized by glycerol. The reaction mechanism of the enzyme was found to be sequential with respect to substrate, and the enzyme formed a hydroxamic acid in the absence of Mg++. The Km for chorismic acid was 1.25 × 10−4m, and the Km for l-glutamine was 5.5 × 10−4m. Enzyme activity was inhibited by tryptophan noncompetitively with respect to chorismic acid and uncompetitively with respect to l-glutamine. An analysis of the inhibition patterns indicated that tryptophan may act as a dead end inhibitor and bind at the catalytic site. Enzyme activity could be completely inhibited in vitro and in vivo under the appropriate conditions, and enzyme synthesis was sensitive to repression by tryptophan. A sedimentation coefficient of 5.5S and an estimated molecular weight of 90,000 were obtained for the enzyme. PMID:5413822

  3. Degenerate Connective Polypeptide 1 (CP1) Domain from Human Mitochondrial Leucyl-tRNA Synthetase*

    PubMed Central

    Ye, Qing; Wang, Meng; Fang, Zhi-Peng; Ruan, Zhi-Rong; Ji, Quan-Quan; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with noncognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain that has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different noncognate amino acids are not completely understood. Here, we identified the essential role of the evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, a broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for noncognate norvaline (Nva) and valine (Val). hmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Moreover, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity and acquired fidelity during aminoacylation. This work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency. PMID:26272616

  4. Holocarboxylase synthetase deficiency pre and post newborn screening.

    PubMed

    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.

  5. Novel insights into regulation of asparagine synthetase in conifers.

    PubMed

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

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

  6. Holocarboxylase synthetase deficiency pre and post newborn screening.

    PubMed

    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. Peptides from aminoacyl-tRNA synthetases can cure the defects due to mutations in mt tRNA genes.

    PubMed

    Francisci, Silvia; Montanari, Arianna; De Luca, Cristina; Frontali, Laura

    2011-11-01

    Recent results from several laboratories have confirmed that human and yeast leucyl- and valyl-tRNA synthetases can rescue the respiratory defects due to mutations in mitochondrial tRNA genes. In this report we show that this effect cannot be ascribed to the catalytic activity per se and that isolated domains of aminoacyl-tRNA synthetases and even short peptides thereof have suppressing effects.

  8. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander; Cropp, Ashton T; Chin, Jason W; Anderson, Christopher J; Schultz, Peter G

    2013-05-21

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  9. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

    2014-08-26

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  10. Unnatural reactive amino acid genetic code additions

    SciTech Connect

    Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

    2011-02-15

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  11. "JCE" Classroom Activity #109: My Acid Can Beat Up Your Acid!

    ERIC Educational Resources Information Center

    Putti, Alice

    2011-01-01

    In this guided-inquiry activity, students investigate the ionization of strong and weak acids. Bead models are used to study acid ionization on a particulate level. Students analyze seven strong and weak acid models and make generalizations about the relationship between acid strength and dissociation. (Contains 1 table and 2 figures.)

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

    PubMed Central

    Avidan, Omri; Pick, Uri

    2015-01-01

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

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

    PubMed

    Avidan, Omri; Pick, Uri

    2015-12-01

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

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

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

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

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

    SciTech Connect

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

    2007-09-01

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

  16. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

    PubMed

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka; Mahfouz, Magdy M

    2015-10-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-10-01

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

  20. Comparison of histidine recognition in human and trypanosomatid histidyl-tRNA synthetases

    PubMed Central

    Koh, Cho Yeow; Wetzel, Allan B; de van der Schueren, Will J.; Hol, Wim G. J.

    2014-01-01

    As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS·His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. Remarkably, the analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS. PMID:25151410

  1. Antioxidant and antimicrobial activities of cinnamic acid derivatives.

    PubMed

    Sova, M

    2012-07-01

    Cinnamic acid is an organic acid occurring naturally in plants that has low toxicity and a broad spectrum of biological activities. In the search for novel pharmacologically active compounds, cinnamic acid derivatives are important and promising compounds with high potential for development into drugs. Many cinnamic acid derivatives, especially those with the phenolic hydroxyl group, are well-known antioxidants and are supposed to have several health benefits due to their strong free radical scavenging properties. It is also well known that cinnamic acid has antimicrobial activity. Cinnamic acid derivatives, both isolated from plant material and synthesized, have been reported to have antibacterial, antiviral and antifungal properties. Acids, esters, amides, hydrazides and related derivatives of cinnamic acid with such activities are here reviewed.

  2. Genetically encoded fluorescent coumarin amino acids

    DOEpatents

    Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

    2012-06-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  3. Genetically encoded fluorescent coumarin amino acids

    DOEpatents

    Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

    2010-10-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

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

    PubMed

    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

  5. Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

    PubMed Central

    Gosling, J. P.; Duggan, P. F.

    1971-01-01

    Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

  6. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  7. Novel subunit-subunit interactions in the structure of glutamine synthetase.

    PubMed

    Almassy, R J; Janson, C A; Hamlin, R; Xuong, N H; Eisenberg, D

    We present an atomic model for glutamine synthetase, an enzyme of central importance in bacterial nitrogen metabolism, from X-ray crystallography. The 12 identical subunits are arranged as the carbon atoms in two face-to-face benzene rings, with unusual subunit contacts. Our model, which places the active sites at the subunit interfaces, suggests a mechanism for the main functional role of glutamine synthetase: how the enzyme regulates the rate of synthesis of glutamine in response to covalent modification and feedback inhibition. PMID:2876389

  8. Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

    PubMed

    Vaishnav, P; Randev, S; Jatiani, S; Aggarwal, S; Keharia, H; Vyas, P R; Nareshkumar, G; Archana, G

    2000-09-01

    Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc). PMID:12561954

  9. Genetics Home Reference: holocarboxylase synthetase deficiency

    MedlinePlus

    ... important for the effective use of biotin, a B vitamin found in foods such as liver, egg yolks, and milk. Holocarboxylase synthetase attaches biotin to certain enzymes that are essential for the normal production and breakdown of proteins, fats, and carbohydrates in ...

  10. Genetics Home Reference: glutathione synthetase deficiency

    MedlinePlus

    ... PubMed Njålsson R. Glutathione synthetase deficiency. Cell Mol Life Sci. 2005 Sep;62(17):1938-45. Review. Citation on PubMed Ristoff E, Larsson A. Inborn errors in the metabolism of glutathione. Orphanet J Rare Dis. 2007 Mar 30;2:16. Review. Citation on PubMed or ...

  11. Fluxless soldering using activated acid vapors

    SciTech Connect

    Frear, D.R.; Keicher, D.M.

    1992-01-01

    Acid vapors have been used to fluxlessly reduce metal oxides and enhance wetting of solder on metallizations. Dilute solutions of hydrogen, acetic acid and formic acid in an inert carrier gas of nitrogen or argon were used with the sessile drop technique for 60Sn-40 Pb solder on Cu and Au/Ni metallizations. The time to reduce metal oxides and the extent of wetting as a function of acid vapor concentrations were characterized. Acetic and formic acids reduce the surface metal oxides sufficiently to form metallurgically sound solder joints. Hydrogen did not reduce oxides rapidly enough at 220{degree}C to be suitable for soldering applications. The optimum conditions for oxide reduction with formic acid was with an acid vapor concentration in nitrogen carrier gas of 4% for Cu metallizations and 1.6% on Au/Ni. The acetic acid vapor concentration, also in nitrogen, was optimized at 1.5% for both metallizations. Above a vapor concentration of 1.5%, the acetic acid combined with the bare metal to form acetates which increased the wetting time. These results indicate that acid vapor fluxless soldering is a viable alternative to traditional flux soldering.

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

    PubMed

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology. PMID:25310879

  13. Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl-tRNA synthetase.

    PubMed

    Prevost, G; Eriani, G; Kern, D; Dirheimer, G; Gangloff, J

    1989-03-15

    Aspartyl-tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lengths from the enzyme C-terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data strongly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, kappa cat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule. Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild-type aminoacylation activity. This involvement of the C-terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.

  14. Tryptophanyl-tRNA synthetase Urzyme: a model to recapitulate molecular evolution and investigate intramolecular complementation.

    PubMed

    Pham, Yen; Kuhlman, Brian; Butterfoss, Glenn L; Hu, Hao; Weinreb, Violetta; Carter, Charles W

    2010-12-01

    We substantiate our preliminary description of the class I tryptophanyl-tRNA synthetase minimal catalytic domain with details of its construction, structure, and steady-state kinetic parameters. Generating that active fragment involved deleting 65% of the contemporary enzyme, including the anticodon-binding domain and connecting peptide 1, CP1, a 74-residue internal segment from within the Rossmann fold. We used protein design (Rosetta), rather than phylogenetic sequence alignments, to identify mutations to compensate for the severe loss of modularity, thus restoring stability, as evidenced by renaturation described previously and by 70-ns molecular dynamics simulations. Sufficient solubility to enable biochemical studies was achieved by expressing the redesigned Urzyme as a maltose-binding protein fusion. Michaelis-Menten kinetic parameters from amino acid activation assays showed that, compared with the native full-length enzyme, TrpRS Urzyme binds ATP with similar affinity. This suggests that neither of the two deleted structural modules has a strong influence on ground-state ATP binding. However, tryptophan has 10(3) lower affinity, and the Urzyme has comparably reduced specificity relative to the related amino acid, tyrosine. Molecular dynamics simulations revealed how CP1 may contribute significantly to cognate amino acid specificity. As class Ia editing domains are nested within the CP1, this finding suggests that this module enhanced amino acid specificity continuously, throughout their evolution. We call this type of reconstructed protein catalyst an Urzyme (Ur prefix indicates original, primitive, or earliest). It establishes a model for recapitulating very early steps in molecular evolution in which fitness may have been enhanced by accumulating entire modules, rather than by discrete amino acid sequence changes. PMID:20864539

  15. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  16. Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus.

    PubMed Central

    Logan, D T; Mazauric, M H; Kern, D; Moras, D

    1995-01-01

    The sequence and crystal structure at 2.75 A resolution of the homodimeric glycyl-tRNA synthetase from Thermus thermophilus, the first representative of the last unknown class II synthetase subgroup, have been determined. The three class II synthetase sequence motifs are present but the structure was essential for identification of motif 1, which does not possess the proline previously believed to be an essential class II invariant. Nevertheless, crucial contacts with the active site of the other monomer involving motif 1 are conserved and a more comprehensive description of class II now becomes possible. Each monomer consists of an active site strongly resembling that of the aspartyl and seryl enzymes, a C-terminal anticodon recognition domain of 100 residues and a third domain unusually inserted between motifs 1 and 2 almost certainly interacting with the acceptor arm of tRNA(Gly). The C-terminal domain has a novel five-stranded parallel-antiparallel beta-sheet structure with three surrounding helices. The active site residues most probably responsible for substrate recognition, in particular in the Gly binding pocket, can be identified by inference from aspartyl-tRNA synthetase due to the conserved nature of the class II active site. Images PMID:7556056

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

    SciTech Connect

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

    1987-10-06

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

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

    PubMed Central

    Roongsawang, Niran; Washio, Kenji; Morikawa, Masaaki

    2011-01-01

    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. PMID:21339982

  19. Physiological activities of hydroxyl fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the search of value-added products from surplus soybean oil, we produced many new hydroxy fatty acids through microbial bioconversion. Hydroxy fatty acids are used in a wide range of industrial products, such as resins, waxes, nylons plastics, lubricants, cosmetics, and additives in coatings and...

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

    PubMed

    Masalkar, Pintu D; Roberts, Daniel M

    2015-01-16

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

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

    SciTech Connect

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

    2008-07-08

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

  2. Acetyl-CoA Synthetase 2 Promotes Acetate Utilization and Maintains Cancer Cell Growth under Metabolic Stress

    PubMed Central

    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

    2015-01-01

    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 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

  3. Physical activity as a determinant of fecal bile acid levels

    PubMed Central

    Wertheim, Betsy C.; Martínez, María Elena; Ashbeck, Erin L.; Roe, Denise J.; Jacobs, Elizabeth T.; Alberts, David S.; Thompson, Patricia A.

    2009-01-01

    Physical activity is protective against colon cancer, whereas colonic bile acid exposure is a suspected risk factor. While likely related, the association between physical activity and bile acid levels has not been well studied. Furthermore, the effect of triglycerides, which are known to modify bile acid levels, on this relationship has not been investigated. We conducted a cross-sectional analysis of baseline fecal bile acid levels for 735 colorectal adenoma formers obtained from participants in a phase III ursodeoxycholic acid chemoprevention trial. Compared to the lowest quartile of recreational physical activity duration, the highest quartile was associated with a 17% lower fecal bile acid concentration, adjusted for age, sex, dietary fiber intake, and body mass index (P = 0.042). Furthermore, consistent with a previously established relationship between serum triglyceride levels and bile acid metabolism, we stratified by triglyceride level and observed a 34% lower fecal bile acid concentration (highest versus lowest quartiles of physical activity) in individuals with low triglycerides (< 136 mg/dL; P = 0.002). In contrast, no association between physical activity and fecal bile acid concentration was observed for subjects with high triglycerides (≥ 136 mg/dL). Our results suggest that the biological mechanism responsible for the protective effect of physical activity on the incidence of colon cancer may be partially mediated by decreasing colonic bile acid exposure. However, this effect may be limited to individuals with lower triglyceride levels. PMID:19383885

  4. Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.

    PubMed

    Taupin, C M; Härtlein, M; Leberman, R

    1997-01-15

    The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

  5. Croconaine rotaxane for acid activated photothermal heating and ratiometric photoacoustic imaging of acidic pH†

    PubMed Central

    Guha, Samit; Shaw, Gillian Karen; Mitcham, Trevor M.; Bouchard, Richard R.

    2015-01-01

    Absorption of 808 nm laser light by liposomes containing a pH sensitive, near-infrared croconaine rotaxane dye increases dramatically in weak acid. A stealth liposome composition permits acid activated, photothermal heating and also acts as an effective nanoparticle probe for ratiometric photoacoustic imaging of acidic pH in deep sample locations, including a living mouse. PMID:26502996

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

    PubMed Central

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

    2015-01-01

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

  7. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    PubMed

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  8. Determinants for tRNA-Dependent Pretransfer Editing in the Synthetic Site of Isoleucyl-tRNA Synthetase

    PubMed Central

    2015-01-01

    The accurate expression of genetic information relies on the fidelity of amino acid–tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs. PMID:25207837

  9. Potentiometric Acid-Base Titrations with Activated Graphite Electrodes

    NASA Astrophysics Data System (ADS)

    Riyazuddin, P.; Devika, D.

    1997-10-01

    Dry cell graphite (DCG) electrodes activated with potassium permanganate are employed as potentiometric indicator electrodes for acid-base titrations. Special attention is given to an indicator probe comprising activated DCG-non-activiated DCG electrode couple. This combination also proves suitable for the titration of strong or weak acids.

  10. The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.

    PubMed

    Thoden, J B; Raushel, F M; Wesenberg, G; Holden, H M

    1999-08-01

    Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain. PMID:10428826

  11. Dissection of the conduit for allosteric control of carbamoyl phosphate synthetase by ornithine.

    PubMed

    Pierrat, Olivier A; Javid-Majd, Farah; Raushel, Frank M

    2002-04-01

    Ornithine is an allosteric activator of carbamoyl phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors. PMID:11913967

  12. Aminoacyl-tRNA synthetases in medicine and disease

    PubMed Central

    Yao, Peng; Fox, Paul L

    2013-01-01

    Aminoacyl-tRNA synthetases (ARSs) are essential and ubiquitous ‘house-keeping’ enzymes responsible for charging amino acids to their cognate tRNAs and providing the substrates for global protein synthesis. Recent studies have revealed a role of multiple ARSs in pathology, and their potential use as pharmacological targets and therapeutic reagents. The ongoing discovery of genetic mutations in human ARSs is increasing exponentially and can be considered an important determinant of disease etiology. Several chemical compounds target bacterial, fungal and human ARSs as antibiotics or disease-targeting medicines. Remarkably, ongoing exploration of noncanonical functions of ARSs has shown important contributions to control of angiogenesis, inflammation, tumourigenesis and other important physiopathological processes. Here, we summarize the roles of ARSs in human diseases and medicine, focusing on the most recent and exciting discoveries. PMID:23427196

  13. Natural cinnamic acids, synthetic derivatives and hybrids with antimicrobial activity.

    PubMed

    Guzman, Juan David

    2014-11-25

    Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC) of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.

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

    SciTech Connect

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

    1987-01-27

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

  15. Palmitate activation by fatty acid transport protein 4 as a model system for hepatocellular apoptosis and steatosis.

    PubMed

    Seeßle, Jessica; Liebisch, Gerhard; Schmitz, Gerd; Stremmel, Wolfgang; Chamulitrat, Walee

    2015-05-01

    Fatty acid transport protein (FATP) 4 is a minor FATP in the liver but it has some activity towards palmitate 16:0 (Pal). We here chose FATP4 as a representative model enzyme for acyl-CoA synthetases (ACSs), and FATPs to determine whether Pal activation would lead to apoptosis and alteration in lipid metabolism. By using FATP4 overexpressed (FATP4) Huh-7 cells, we showed that FATP4 was localized in the endoplasmic reticulum (ER) and mitochondria of FATP4 cells. FATP4 cells were more responsive to Pal than the control GFP cells in increasing palmitoyl-CoA and oleoyl-CoA activities as well as apoptosis by ~2-3 folds. The lipoapoptosis susceptibility by FATP4 was coupled with the increased JNK, PUMA, caspase3, PARP-1 activation as well as Rac-1-mediated cytoskeletal reorganization, and decreased insulin sensitivity. This was associated with increased contents of neutral lipids and significant alteration in composition of phospholipids and sphingolipids including increased lysophosphatidylcholine (LPC), ceramide, and hexosylceramide, as well as an increase of saturated:polyunsaturated fatty acid ratio in LPC and PC, but a decrease of this ratio in phosphatidylethanolamine pool. By use of ceramide synthase inhibitors, our results showed that FATP4-sensitized lipoapoptosis was not mediated by ceramides. Moreover, FATP4 expression was increased in fatty livers in vivo. Thus, our model system has provided a clue that Pal activation FATP4 triggers hepatocellular apoptosis via altered phospholipid composition and steatosis by acylation into complex lipids. This may be a redundant mechanism for other ER-localizing ACSs and FATPs in the liver, and hence their involvement in the development of fatty liver disease.

  16. [Biological activity of retinoic acid and methylretinoate].

    PubMed

    Dusheĭko, A A; Chernukhina, L A; Blazhevich, M A; Davydova, L P

    1980-01-01

    Vitamin A lack in the diet of chicken produces a significant increase in the glandular stomach as well as formation of erosions and ulcers on the surface of the mucous membrane of the intermediate zone. Replacement of retinyl acetate in the diet by retinoic acid or methyl retionate gives no rise to changes in the morphological integrity of the glandular stomach of the chickens. Moreover, these compounds produce a reverse development of vitamin A-induced changes. It is thus concluded that when the diet lacks vitamin A, both retinoic acid and methyl retionate are capable of maintaining the structural integrity of the stomach.

  17. Cloning and characterization of the ferulic acid catabolic genes of Sphingomonas paucimobilis SYK-6.

    PubMed

    Masai, Eiji; Harada, Kyo; Peng, Xue; Kitayama, Hirotaka; Katayama, Yoshihiro; Fukuda, Masao

    2002-09-01

    Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase

  18. Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation.

    PubMed

    McClure, P R; Coker, G T; Schubert, K R

    1983-03-01

    Detached roots and nodules of the N(2)-fixing species, Albus glutinosa (European black alder), actively assimilate CO(2). The maximum rates of dark CO(2) fixation observed for detached nodules and roots were 15 and 3 micromoles CO(2) fixed per gram dry weight per hour, respectively. The net incorporation of CO(2) in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO(2) fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO(2) fixation and N(2) fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total (14)C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N(2)-fixing nodules and roots of alder. PMID:16662882

  19. Perfluoroalkyl acids : Recent activities and research progress

    EPA Science Inventory

    The perfluoroalkyl acids (PFAAs) are a family of man-made fluorinated organic chemicals consisting of a carbon backbone typically of four to fourteen in length and a charged functional moiety (primarily carboxylate, sulfonate or phosphonate). The two most widely known PFAAs are ...

  20. A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves.

    PubMed

    Kamachi, K; Yamaya, T; Mae, T; Ojima, K

    1991-06-01

    Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants. PMID:16668201

  1. Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei.

    PubMed

    Katoh, Hiroki; Tamaki, Hideyuki; Tokutake, Yuka; Hanada, Satoshi; Chohnan, Shigeru

    2013-04-01

    Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

  2. Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

    PubMed

    Fuentes, S I; Allen, D J; Ortiz-Lopez, A; Hernández, G

    2001-05-01

    Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution. PMID:11432923

  3. Novel nonribosomal peptide synthetase (NRPS) genes sequenced from intertidal mudflat bacteria.

    PubMed

    Tambadou, Fatoumata; Lanneluc, Isabelle; Sablé, Sophie; Klein, Géraldine L; Doghri, Ibtissem; Sopéna, Valérie; Didelot, Sandrine; Barthélémy, Cyrille; Thiéry, Valérie; Chevrot, Romain

    2014-08-01

    Nonribosomal peptide synthetases (NRPS) are actively sought out, due to pharmacologically important activities of their metabolites. In marine environment, the most prevalent nonribosomal peptide antibiotic producers are sponges inhabiting microorganisms. Conversely, strains from marine sediments and more especially from intertidal mudflats have not been extensively screened for the presence of new NRPS. In this study, for the first time, a collection of one hundred intertidal mudflat bacterial isolates (Marennes-Oléron Bay, France) was assessed for (1) the presence of NRPS genes by degenerated PCR targeting conserved adenylation domains and (2) for their production of antimicrobial molecules. (1) Bacteria with adenylation domains (14 strains) were identified by 16S rRNA gene sequence analysis and grouped into Firmicutes (one strain) and Proteobacteria (13 strains). In silico analysis of the NRPS amino acid sequences (n = 7) showed 41-58% ID with sequences found in the NCBI database. Three new putative adenylation domain signatures were found. (2) The culture supernatant of one of these strains, identified as a Bacillus, was shown to strongly inhibit the growth of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. This study portends that the intertidal mudflat niche could be of interest for the discovery of new NRPS genes and antimicrobial producing strains. PMID:25039651

  4. Role of Jo-1 in the Immunopathogenesis of the Anti-synthetase Syndrome.

    PubMed

    Ascherman, Dana P

    2015-09-01

    Histidyl-tRNA synthetase (HRS = Jo-1) represents a key autoantibody target in the anti-synthetase syndrome that is marked by myositis as well as extra-muscular organ complications including interstitial lung disease (ILD). Over the last 25 years, a wealth of clinical, epidemiological, genetic, and experimental data have collectively supported a role for Jo-1 in mediating deleterious cell-mediated, adaptive immune responses contributing to the disease phenotype of the anti-synthetase syndrome. Complementing these studies, more recent work suggests that unique, non-enzymatic functional properties of Jo-1 also endow this antigen with the capacity to activate components of the innate immune system, particularly cell surface as well as endosomal Toll-like receptors and their downstream signaling pathways. Combining these facets of Jo-1-mediated immunity now supports a more integrated model of disease pathogenesis that should lead to improved therapeutic targeting in the anti-synthetase syndrome and related subsets of idiopathic inflammatory myopathy.

  5. Prostaglandin synthetase and prostacyclin synthetase in mature rat skeletal muscles: immunohistochemical localisation to arterioles, tendons and connective tissues.

    PubMed Central

    McLennan, I S; Macdonald, R E

    1991-01-01

    Mature skeletal muscles produce appreciable quantities of prostacyclin (PGI2) and smaller amounts of PGF2 alpha and PGE2, but the sources of these prostaglandins within skeletal muscle are unknown. Monoclonal antibodies to prostaglandin synthetase and prostacyclin synthetase were used to determine which muscle cells produce prostaglandins. The antibody to prostacyclin synthetase stained the tendon, fascia, epimysium and the arteries leading to the muscles. The endothelia of arterioles were also stained in the tibialis anterior and cremaster but not in the soleus muscles. Only trace levels of immunoreactivity were observed with the antibody to prostaglandin synthetase in normal muscles. However, immunoreactivity was observed in the muscles of rats that had been pretreated with aspirin, a drug that inhibits and stabilises prostaglandin synthetase. In muscles of the aspirin-treated rats, all cell types that were stained by the antiprostacyclin synthetase also reacted weakly with the antibody to prostaglandin synthetase. In addition, some cells in the endomysium were strongly stained with the antiprostaglandin synthetase but not with the antiprostacyclin synthetase. We conclude that (1) at least one aspect of the regulation of blood flow in the microcirculation of slow muscles is different from that of fast muscles, (2) that the tendon and connective tissue is the major source of PGI2 in mature skeletal muscles, and (3) that the prostaglandin-dependent effects of insulin and some other stimuli on skeletal muscle may be mediated by the muscle's arterioles or connective tissue. Images Fig. 1 Fig. 2 Fig. 3 PMID:1810931

  6. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    PubMed Central

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  7. Surface-active properties of humic and sulfochlorohumic acids

    SciTech Connect

    Ryabova, I.N.; Mustafina, G.A.; Akkulova, Z.G.; Satymbaeva, A.S.

    2009-10-15

    The surface tension of alkaline solutions of humic acids and their sulfochloroderivatives, which are synthesized by sulfonation of chlorohumic acids isolated from coal chlorinated by the electrochemical method, is investigated. It is established that humic compounds possess weak surface activity. Basic adsorption parameters are calculated.

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

    DOE PAGES

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

    2014-01-01

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

  9. Synthesis and evaluation of dioleoyl glyceric acids showing antitrypsin activity.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Sato, Shun; Kitamoto, Dai; Sakaki, Keiji

    2011-01-01

    Previously, Lešová et al. reported the isolation and identification of metabolite OR-1, showing antitrypsin activity, produced during fermentation by Penicillium funiculosum. The structure of OR-1 was a mixture of glyceric acid (GA), esterified with C(14)-C(18) fatty acids, and oleic acid (C18:1) as the most predominant fatty acid (Folia Microbiol. 46, 21-23, 2001). In this study, dioleoyl D-GA and dioleoyl L-GA were synthesized via diesterification with oleoyl chloride, and their antitrypsin activities were evaluated using both a disk diffusion method and spectral absorption measurements. The results show that both compounds and their equivalent mixtures possess antitrypsin activities; however, their IC(50) values (approximately 2 mM) are much higher than that of OR-1 (4.25 µM), suggesting that dioleoyl GA does not play a major role in the OR-1 antitrypsin activity. PMID:21606621

  10. Multiple forms of acid phosphatase activity in Gaucher's disease.

    PubMed

    Chambers, J P; Peters, S P; Glew, R H; Lee, R E; McCafferty, L R; Mercer, D W; Wenger, D A

    1978-07-01

    Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to

  11. Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.

    PubMed

    Katavic, Vesna; Mietkiewska, Elzbieta; Barton, Dennis L; Giblin, E Michael; Reed, Darwin W; Taylor, David C

    2002-11-01

    Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.

  12. Expression of Efflux Pumps and Fatty Acid Activator One Genes in Azole Resistant Candida Glabrata Isolated From Immunocompromised Patients.

    PubMed

    Farahyar, Shirin; Zaini, Farideh; Kordbacheh, Parivash; Rezaie, Sassan; Falahati, Mehraban; Safara, Mahin; Raoofian, Reza; Hatami, Kamran; Mohebbi, Masoumeh; Heidari, Mansour

    2016-07-01

    Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology, semi-quantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (≥2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates ≥2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates. PMID:27424018

  13. Activation of Inactive Nitrogenase by Acid-Treated Component I

    PubMed Central

    Nagatani, H. H.; Shah, Vinod K.; Brill, Winston J.

    1974-01-01

    When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N2. Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N2-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum. PMID:4218230

  14. Synthesis and antituberculosis activity of new fatty acid amides.

    PubMed

    D'Oca, Caroline Da Ros Montes; Coelho, Tatiane; Marinho, Tamara Germani; Hack, Carolina Rosa Lopes; Duarte, Rodrigo da Costa; da Silva, Pedro Almeida; D'Oca, Marcelo Gonçalves Montes

    2010-09-01

    This work reports the synthesis of new fatty acid amides from C16:0, 18:0, 18:1, 18:1 (OH), and 18:2 fatty acids families with cyclic and acyclic amines and demonstrate for the first time the activity of these compounds as antituberculosis agents against Mycobacterium tuberculosis H(37)Rv, M. tuberculosis rifampicin resistance (ATCC 35338), and M. tuberculosis isoniazid resistance (ATCC 35822). The fatty acid amides derivate from ricinoleic acid were the most potent one among a series of tested compounds, with a MIC 6.25 microg/mL for resistance strains.

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

    PubMed

    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

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

    PubMed Central

    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

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

    PubMed

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

    2016-05-03

    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.

  18. Structural Requirements for the Procoagulant Activity of Nucleic Acids

    PubMed Central

    Gansler, Julia; Jaax, Miriam; Leiting, Silke; Appel, Bettina; Greinacher, Andreas; Fischer, Silvia; Preissner, Klaus T.

    2012-01-01

    Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects. PMID:23226277

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

    PubMed

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

    2015-08-14

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

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

    PubMed Central

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

    2015-01-01

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans. PMID:26271225

  1. Inhibition of Plasmodium falciparum dihydropteroate synthetase and growth in vitro by sulfa drugs.

    PubMed Central

    Zhang, Y; Meshnick, S R

    1991-01-01

    The Michaelis-Menten inhibitory constants (Kis) and the concentrations required for 50% inhibition of the Plasmodium falciparum dihydropteroate synthetase were determined for six sulfa drugs. These drugs inhibited the in vitro growth of P. falciparum (50% lethal concentration) at concentrations of 30 to 500 nM; these concentrations were 100 to 1,000 times lower than the concentrations required for 50% inhibition and Kis (6 to 500 microM). The uptake of p-aminobenzoic acid was not inhibited by the sulfa drugs. However, infected erythrocytes took up more labeled sulfamethoxazole than did uninfected erythrocytes. Thus, the concentration of sulfa drugs by malaria parasites may explain how sulfa drugs inhibit in vitro growth of parasites through the inhibition of dihydropteroate synthetase. PMID:2024960

  2. GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase*

    PubMed Central

    Bunpo, Piyawan; Dudley, Allison; Cundiff, Judy K.; Cavener, Douglas R.; Wek, Ronald C.; Anthony, Tracy G.

    2009-01-01

    Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1. PMID:19783659

  3. Antiproliferative activity of synthetic fatty acid amides from renewable resources.

    PubMed

    dos Santos, Daiane S; Piovesan, Luciana A; D'Oca, Caroline R Montes; Hack, Carolina R Lopes; Treptow, Tamara G M; Rodrigues, Marieli O; Vendramini-Costa, Débora B; Ruiz, Ana Lucia T G; de Carvalho, João Ernesto; D'Oca, Marcelo G Montes

    2015-01-15

    In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line-the most aggressive CNS cancer.

  4. Studies on the control of hexosamine biosynthesis by glucosamine synthetase

    PubMed Central

    Winterburn, P. J.; Phelps, C. F.

    1971-01-01

    1. The nature of the feedback inhibition of hexosamine biosynthesis on rat liver glucosamine synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) by UDP-N-acetylglucosamine was investigated in detail. 2. Further modifiers of physiological importance are described. Glucose 6-phosphate and AMP potentiated the UDP-N-acetylglucosamine inhibition, and UTP behaved as an activator. These three compounds only exerted their action when the feedback inhibitor was bound to the enzyme. 3. ATP also inhibited the enzyme. 4. The actions of these various effectors are discussed in kinetic terms. 5. An interpretation of these findings with reference to the regulation of hexosamine biosynthesis is presented. PMID:5114979

  5. Novel Bioactivity of Ellagic Acid in Inhibiting Human Platelet Activation

    PubMed Central

    Chang, Yi; Chen, Wei-Fan; Lin, Kuan-Hung; Hsieh, Cheng-Ying; Chou, Duen-Suey; Lin, Li-Jyun; Sheu, Joen-Rong; Chang, Chao-Chien

    2013-01-01

    Pomegranates are widely consumed either as fresh fruit or in beverage form as juice and wine. Ellagic acid possesses potent antioxidative properties; it is known to be an effective phytotherapeutic agent with antimutagenic and anticarcinogenic qualities. Ellagic acid (20 to 80 μM) exhibited a potent activity in inhibiting platelet aggregation stimulated by collagen; however, it did not inhibit platelet aggregation stimulated by thrombin, arachidonic acid, or U46619. Treatment with ellagic acid (50 and 80 μM) significantly inhibited platelet activation stimulated by collagen; this alteration was accompanied by the inhibition of relative [Ca2+]i mobilization, and the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt, as well as hydroxyl radical (OH●) formation. In addition, ellagic acid also inhibited p38 MAPK and Akt phosphorylation stimulated by hydrogen peroxide. By contrast, ellagic acid did not significantly affect PKC activation and platelet aggregation stimulated by PDBu. This study is the first to show that, in addition to being considered a possible agent for preventing tumor growth, ellagic acid possesses potent antiplatelet properties. It appears to initially inhibit the PLCγ2-PKC cascade and/or hydroxyl radical formation, followed by decreased phosphorylation of MAPKs and Akt, ultimately inhibiting platelet aggregation. PMID:23533502

  6. Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

    PubMed

    Zaki, Ahmed; Ashour, Ahmed; Mira, Amira; Kishikawa, Asuka; Nakagawa, Toshinori; Zhu, Qinchang; Shimizu, Kuniyoshi

    2016-05-01

    Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26887328

  7. Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

    PubMed

    Zaki, Ahmed; Ashour, Ahmed; Mira, Amira; Kishikawa, Asuka; Nakagawa, Toshinori; Zhu, Qinchang; Shimizu, Kuniyoshi

    2016-05-01

    Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    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 (Acsl1T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1T−/− hearts, control and Acsl1T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function. PMID:26136511

  11. Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis

    PubMed Central

    de Cima, Sergio; Polo, Luis M.; Díez-Fernández, Carmen; Martínez, Ana I.; Cervera, Javier; Fita, Ignacio; Rubio, Vicente

    2015-01-01

    Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. PMID:26592762

  12. Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis.

    PubMed

    de Cima, Sergio; Polo, Luis M; Díez-Fernández, Carmen; Martínez, Ana I; Cervera, Javier; Fita, Ignacio; Rubio, Vicente

    2015-01-01

    Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. PMID:26592762

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

    PubMed Central

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

    1999-01-01

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

  14. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  15. Retinal Vasculitis in Anti-Synthetase Syndrome.

    PubMed

    Donovan, Christopher P; Pecen, Paula E; Baynes, Kimberly; Ehlers, Justis P; Srivastava, Sunil K

    2016-09-01

    A 31-year-old woman with a history of anti-synthetase syndrome-related myositis and interstitial lung disease presented with acute-onset blurry vision and rash on her hands and feet. Visual acuity was hand motion in her right eye and 20/40 in her left eye. Dilated fundus exam showed extensive retinal vasculitis, diffuse intraretinal hemorrhages, and subretinal fluid. Optical coherence tomography revealed significant macular thickening, and fluorescein angiography revealed vascular leakage with peripheral nonperfusion. Aggressive systemic immunosuppression was initiated, with gradual resolution of her disease during 8 months of follow-up. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:874-879.].

  16. Retinal Vasculitis in Anti-Synthetase Syndrome.

    PubMed

    Donovan, Christopher P; Pecen, Paula E; Baynes, Kimberly; Ehlers, Justis P; Srivastava, Sunil K

    2016-09-01

    A 31-year-old woman with a history of anti-synthetase syndrome-related myositis and interstitial lung disease presented with acute-onset blurry vision and rash on her hands and feet. Visual acuity was hand motion in her right eye and 20/40 in her left eye. Dilated fundus exam showed extensive retinal vasculitis, diffuse intraretinal hemorrhages, and subretinal fluid. Optical coherence tomography revealed significant macular thickening, and fluorescein angiography revealed vascular leakage with peripheral nonperfusion. Aggressive systemic immunosuppression was initiated, with gradual resolution of her disease during 8 months of follow-up. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:874-879.]. PMID:27631486

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development. PMID:26307137

  19. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    SciTech Connect

    Rubin, Edward M.

    1980-01-01

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

  20. Activity of earthworm in Latosol under simulated acid rain stress.

    PubMed

    Zhang, Jia-En; Yu, Jiayu; Ouyang, Ying

    2015-01-01

    Acid rain is still an issue of environmental concerns. This study investigated the impacts of simulated acid rain (SAR) upon earthworm activity from the Latosol (acidic red soil). Laboratory experiment was performed by leaching the soil columns grown with earthworms (Eisenia fetida) at the SAR pH levels ranged from 2.0 to 6.5 over a 34-day period. Results showed that earthworms tended to escape from the soil and eventually died for the SAR at pH = 2.0 as a result of acid toxicity. The catalase activity in the earthworms decreased with the SAR pH levels, whereas the superoxide dismutases activity in the earthworms showed a fluctuate pattern: decreasing from pH 6.5 to 5.0 and increasing from pH 5.0 to 4.0. Results implied that the growth of earthworms was retarded at the SAR pH ≤ 3.0.

  1. Spectroscopic studies on the antioxidant activity of ellagic acid

    NASA Astrophysics Data System (ADS)

    Kilic, Ismail; Yeşiloğlu, Yeşim; Bayrak, Yüksel

    2014-09-01

    Ellagic acid (EA, C14H6O8) is a natural dietary polyphenol whose benefits in a variety of diseases shown in epidemiological and experimental studies involve anti-inflammation, anti-proliferation, anti-angiogenesis, anticarcinogenesis and anti-oxidation properties. In vitro radical scavenging and antioxidant capacity of EA were clarified using different analytical methodologies such as total antioxidant activity determination by ferric thiocyanate, hydrogen peroxide scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH) scavenging, 2,2‧-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and superoxide anion radical scavenging, ferrous ions (Fe2+) chelating activity and ferric ions (Fe3+) reducing ability. EA inhibited 71.2% lipid peroxidation of a linoleic acid emulsion at 45 μg/mL concentration. On the other hand, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), α-tocopherol and ascorbic acid displayed 69.8%, 66.8%, 64.5% and 59.7% inhibition on the peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, EA had an effective DPPH• scavenging, ABTSrad + scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe3+) reducing power and ferrous ions (Fe2+) chelating activities. Also, those various antioxidant activities were compared to BHA, BHT, α-tocopherol and ascorbic acid as references antioxidant compounds. These results suggested that EA can be used in the pharmacological, food industry and medicine because of these properties.

  2. Regulatory changes in the control of carbamoyl phosphate synthetase induced by truncation and mutagenesis of the allosteric binding domain.

    PubMed

    Czerwinski, R M; Mareya, S M; Raushel, F M

    1995-10-24

    Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7577987

  3. Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase.

    PubMed

    Evilia, Caryn; Hou, Ya-Ming

    2006-06-01

    Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.

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

    SciTech Connect

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

    1994-09-01

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

  5. Synthesis of Glu-tRNA(Gln) by engineered and natural aminoacyl-tRNA synthetases.

    PubMed

    Rodríguez-Hernández, Annia; Bhaskaran, Hari; Hadd, Andrew; Perona, John J

    2010-08-10

    A protein engineering approach to delineating which distinct elements of homologous tRNA synthetase architectures are responsible for divergent RNA-amino acid pairing specificities is described. Previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) were replaced with the corresponding residues of human glutamyl-tRNA synthetase (GluRS). The engineered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNA(Gln) more than 10(4)-fold more efficiently than GlnRS. Detailed comparison of kinetic parameters between GlnRS S1/L1/L2 and the naturally occurring Methanothermobacter thermautotrophicus GluRS(ND), which is also capable of Glu-tRNA(Gln) synthesis, now shows that both k(cat) and K(m) for glutamate are recapitulated in the engineered enzyme, but that K(m) for tRNA is 200-fold higher. Thus, the simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. We infer that the GlnRS architecture has differentiated to match only cognate amino acid-RNA pairs, and that the substrate selection functions do not operate independently of each other. Design and characterization of four additional hybrids identify further residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites. The robust catalytic function demonstrated in this engineered system offers a novel platform for exploring the stereochemical origins of coding as a property of the ancient Rossmann fold.

  6. Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

    PubMed Central

    Ortega, Jose Luis; Temple, Stephen J.; Bagga, Suman; Ghoshroy, Soumitra; Sengupta-Gopalan, Champa

    2013-01-01

    Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants. PMID:15197594

  7. A Recurrent Loss-of-Function Alanyl-tRNA Synthetase (AARS) Mutation in Patients with Charcot-Marie-Tooth Disease Type 2N (CMT2N)

    PubMed Central

    McLaughlin, Heather M.; Sakaguchi, Reiko; Giblin, William; Wilson, Thomas E.; Biesecker, Leslie; Lupski, James R.; Talbot, Kevin; Vance, Jeffery M.; Züchner, Stephan; Lee, Yi-Chung; Kennerson, Marina; Hou, Ya-Ming; Nicholson, Garth; Antonellis, Anthony

    2011-01-01

    Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation. PMID:22009580

  8. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity

    PubMed Central

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M.; Park, Jin-Byung

    2016-01-01

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass. PMID:27681369

  9. The antimicrobial activities of the cinnamaldehyde adducts with amino acids.

    PubMed

    Wei, Qing-Yi; Xiong, Jia-Jun; Jiang, Hong; Zhang, Chao; Wen Ye

    2011-11-01

    Cinnamaldehyde is a well-established natural antimicrobial compound. It is probable for cinnamaldehyde to react with amino acid forming Schiff base adduct in real food system. In this paper, 9 such kind of adducts were prepared by the direct reaction of amino acids with cinnamaldehyde at room temperature. Their antimicrobial activities against Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were evaluated with benzoic acid as a reference. The adducts showed a dose-dependent activities against the three microbial strains. Both cinnamaldehyde and their adducts were more active against B. subtilis than on E. coli, and their antimicrobial activities were higher at lower pH. Both cinnamaldehyde and its adducts were more active than benzoic acid at the same conditions. The adduct compound A was non-toxic by primary oral acute toxicity study in mice. However, in situ effect of the adduct compound A against E. coli was a little lower than cinnamaldehyde in fish meat. This paper for the first time showed that the cinnamaldehyde adducts with amino acids had similar strong antimicrobial activities as cinnamaldehyde, which may provide alternatives to cinnamaldehyde in food to avoid the strong unacceptable odor of cinnamaldehyde. PMID:21856030

  10. Catalytic Ethanol Dehydration over Different Acid-activated Montmorillonite Clays.

    PubMed

    Krutpijit, Chadaporn; Jongsomjit, Bunjerd

    2016-01-01

    In the present study, the catalytic dehydration of ethanol to obtain ethylene over montmorillonite clays (MMT) with mineral acid activation including H2SO4 (SA-MMT), HCl (HA-MMT) and HNO3 (NA-MMT) was investigated at temperature range of 200 to 400°C. It revealed that HA-MMT exhibited the highest catalytic activity. Ethanol conversion and ethylene selectivity were found to increase with increased reaction temperature. At 400°C, the HA-MMT yielded 82% of ethanol conversion having 78% of ethylene yield. At lower temperature (i.e. 200 to 300°C), diethyl ether (DEE) was a major product. The highest activity obtained from HA-MMT can be attributed to an increase of weak acid sites and acid density by the activation of MMT with HCl. It can be also proven by various characterization techniques that in most case, the main structure of MMT did not alter by acid activation (excepted for NA-MMT). Upon the stability test for 72 h during the reaction, the MMT and HA-MMT showed only slight deactivation due to carbon deposition. Hence, the acid activation of MMT by HCl is promising to enhance the catalytic dehydration of ethanol. PMID:27041515

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

    SciTech Connect

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

    2013-02-08

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

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

    PubMed Central

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

    2015-01-01

    Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNAGln and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca2+ and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC. PMID:26324710

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

    SciTech Connect

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

    1987-08-25

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

  14. Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

    PubMed Central

    Guerreiro, Juliano R.; Lameu, Claudiana; Oliveira, Eduardo F.; Klitzke, Clécio F.; Melo, Robson L.; Linares, Edlaine; Augusto, Ohara; Fox, Jay W.; Lebrun, Ivo; Serrano, Solange M. T.; Camargo, Antonio C. M.

    2009-01-01

    Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases. PMID:19491403

  15. Antileishmanial activity of diterpene acids in copaiba oil

    PubMed Central

    dos Santos, Adriana Oliveira; Izumi, Erika; Ueda-Nakamura, Tânia; Dias-Filho, Benedito Prado; da Veiga-Júnior, Valdir Florêncio; Nakamura, Celso Vataru

    2013-01-01

    Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs. PMID:23440116

  16. The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes.

    PubMed

    Yang, Darren; Pornpattananangkul, Dissaya; Nakatsuji, Teruaki; Chan, Michael; Carson, Dennis; Huang, Chun-Ming; Zhang, Liangfang

    2009-10-01

    This study evaluated the antimicrobial activity of lauric acid (LA) and its liposomal derivatives against Propionibacterium acnes (P. acnes), the bacterium that promotes inflammatory acne. First, the antimicrobial study of three free fatty acids (lauric acid, palmitic acid and oleic acid) demonstrated that LA gives the strongest bactericidal activity against P. acnes. However, a setback of using LA as a potential treatment for inflammatory acne is its poor water solubility. Then the LA was incorporated into a liposome formulation to aid its delivery to P. acnes. It was demonstrated that the antimicrobial activity of LA was not only well maintained in its liposomal derivatives but also enhanced at low LA concentration. In addition, the antimicrobial activity of LA-loaded liposomes (LipoLA) mainly depended on the LA loading concentration per single liposomes. Further study found that the LipoLA could fuse with the membranes of P. acnes and release the carried LA directly into the bacterial membranes, thereby killing the bacteria effectively. Since LA is a natural compound that is the main acid in coconut oil and also resides in human breast milk and liposomes have been successfully and widely applied as a drug delivery vehicle in the clinic, the LipoLA developed in this work holds great potential of becoming an innate, safe and effective therapeutic medication for acne vulgaris and other P. acnes associated diseases. PMID:19665786

  17. Urease inhibitory activities of β-boswellic acid derivatives

    PubMed Central

    2013-01-01

    Background and the purpose of the study Boswellia carterii have been used in traditional medicine for many years for management different gastrointestinal disorders. In this study, we wish to report urease inhibitory activity of four isolated compound of boswellic acid derivative. Methods 4 pentacyclic triterpenoid acids were isolated from Boswellia carterii and identified by NMR and Mass spectroscopic analysis (compounds 1, 3-O-acetyl-9,11-dehydro-β-boswellic acid; 2, 3-O-acetyl-11-hydroxy-β-boswellic acid; 3. 3-O- acetyl-11-keto-β-boswellic acid and 4, 11-keto-β-boswellic acid. Their inhibitory activity on Jack bean urease were evaluated. Docking and pharmacophore analysis using AutoDock 4.2 and Ligandscout 3.03 programs were also performed to explain possible mechanism of interaction between isolated compounds and urease enzyme. Results It was found that compound 1 has the strongest inhibitory activity against Jack bean urease (IC50 = 6.27 ± 0.03 μM), compared with thiourea as a sta