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Sample records for fatty acyl-coa synthetase

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

    PubMed Central

    Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

    2013-01-01

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

  3. A NOVEL 78-KDA FATTY ACYL-COA SYNTHETASE (ACS1) OF BABESIA BOVIS STIMULATES MEMORY CD4+ T LYMPHOCYTE RESPONSES IN B. BOVIS-IMMUNE CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigen-specific CD4+ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4+ T cells from immune cattle. Fract...

  4. EXPRESSION OF TURKEY TRANSCRIPTION FACTORS AND ACYL COA OXIDASE IN DIFFERENT TISSUES AND GENETIC POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several transcription factors are involved in regulating lipid metabolism in various animal tissues. Peroxisome proliferator activated receptor (PPAR) gamma and PPAR alpha regulate both lipogenesis and fatty acid oxidation. Gene fragments for PPAR gamma, PPAR alpha, and acyl CoA oxidase (ACO) have b...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

    PubMed Central

    Jiang, D W; Englund, P T

    2001-01-01

    As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

  7. BROWN ADIPOSE TISSUE FUNCTION IN SHORT-CHAIN ACYL-COA DEHYDROGENASE DEFICIENT MICE

    PubMed Central

    Skilling, Helen; Coen, Paul M.; Fairfull, Liane; Ferrell, Robert E.; Goodpaster, Bret H.; Vockley, Jerry; Goetzman, Eric S.

    2010-01-01

    Brown adipose tissue is a highly specialized organ that uses mitochondrial fatty acid oxidation to fuel nonshivering thermogenesis. In mice, mutations in the acyl-CoA dehydrogenase family of fatty acid oxidation genes are associated with sensitivity to cold. Brown adipose tissue function has not previously been characterized in these knockout strains. Short-chain acyl-CoA dehydrogenase (SCAD) deficient mice were found to have increased brown adipose tissue mass as well as modest cardiac hypertrophy. Uncoupling protein-1 was reduced by 70% in brown adipose tissue and this was not due to a change in mitochondrial number, nor was it due to decreased signal transduction through protein kinase A which is known to be a major regulator of uncoupling protein-1 expression. PKA activity and in vitro lipolysis were normal in brown adipose tissue, although in white adipose tissue a modest increase in basal lipolysis was seen in SCAD−/ − mice. Finally, an in vivo norepinephrine challenge of brown adipose tissue thermogenesis revealed normal heat production in SCAD−/− mice. These results suggest that reduced brown adipose tissue function is not the major factor causing cold sensitivity in acyl-CoA dehydrogenase knockout strains. We speculate that other mechanisms such as shivering capacity, cardiac function, and reduced hepatic glycogen stores are involved. PMID:20727852

  8. Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity.

    PubMed

    Füllekrug, Joachim; Poppelreuther, Margarete

    2016-01-01

    Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The implication of ACS enzymes in widespread lipid associated diseases like type 2 diabetes has rekindled interest in this protein family. Here, we describe in detail how to measure long-chain fatty acyl-CoA synthetase activity by a straightforward radiometric assay. Cell lysates are incubated with ATP, coenzyme A, Mg(2+), and radiolabeled fatty acid bound to BSA. Differential phase partitioning of fatty acids and acyl-CoAs is exploited to quantify the amount of generated acyl-CoA by scintillation counting. The high sensitivity of this assay also allows the analysis of small samples like patient biopsies. PMID:26552674

  9. Control of the Synthesis of Fatty-Acid Synthetase in Rat Liver by Insulin, Glucagon, and Adenosine 3′:5′ Cyclic Monophosphate

    PubMed Central

    Lakshmanan, M. R.; Nepokroeff, Carl M.; Porter, John W.

    1972-01-01

    The usual increase in the activity of liver fatty-acid synthetase that occurs on refeeding of a fat-free diet to previously fasted rats is abolished in diabetic animals. Insulin specifically restores this increase by enhancement of the rate of synthesis of fatty-acid synthetase. However, glucagon and cyclic AMP inhibit the increase in the activity of fatty-acid synthetase. Therefore, the concentration of fatty-acid synthetase in rat liver is under the control of the relative concentrations of insulin and glucagon. PMID:4345502

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

    PubMed Central

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

    1993-01-01

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

  11. Differential Gene Expression and Protein Localization of Cryptosporidium parvum Fatty Acyl-CoA Synthetase Isoforms.

    PubMed

    Guo, Fengguang; Zhang, Haili; Payne, Harold Ross; Zhu, Guan

    2016-03-01

    Cryptosporidium parvum is unable to synthesize fatty acids de novo, but possesses three long-chain fatty acyl-CoA synthetase (CpACS) isoforms for activating fatty acids. We have recently shown that these enzymes could be targeted to kill the parasite in vitro and in vivo. Here, we demonstrated that the CpACS genes were differentially expressed during the parasite life cycle, and their proteins were localized to different subcellular structures by immunofluorescence and immuno-electron microscopies. Among them, CpACS1 displayed as an apical protein in sporozoites and merozoites, but no or little presence during the intracellular merogony until the release of merozoites, suggesting that CpACS1 probably functioned mainly during the parasite invasion and/or early stage of intracellular development. Both CpACS2 and CpACS3 proteins were present in all parasite life cycle stages, in which CpACS2 was present in the parasite and the parasitophorous vacuole membranes (PVM), whereas CpACS3 was mainly present in the parasite plasma membranes with little presence in the PVM. These observations suggest that CpACS2 and CpACS3 may participate in scavenging and transport of fatty acids across the PVM and the parasite cytoplasmic membranes, respectively. PMID:26411755

  12. 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

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

    PubMed Central

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

    1998-01-01

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

  14. Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

    PubMed

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

    1992-07-01

    A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis. PMID:1514693

  15. 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

  16. Elucidating the Pseudomonas aeruginosa fatty acid degradation pathway: identification of additional fatty acyl-CoA synthetase homologues.

    PubMed

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

    2013-01-01

    The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h. PMID:23737986

  17. 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

  18. The Role of Pyruvate Dehydrogenase and Acetyl-Coenzyme A Synthetase in Fatty Acid Synthesis in Developing Arabidopsis Seeds1

    PubMed Central

    Ke, Jinshan; Behal, Robert H.; Back, Stephanie L.; Nikolau, Basil J.; Wurtele, Eve Syrkin; Oliver, David J.

    2000-01-01

    Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1α- and ptE1β-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1β mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1β mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds. PMID:10859180

  19. 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

  20. Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

    PubMed Central

    Jiang, Yanfang; Morgan-Kiss, Rachael M.; Campbell, John W.; Chan, Chi Ho; Cronan, John E.

    2010-01-01

    Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously-supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function. PMID:20028080

  1. Identification of a Long-Chain Polyunsaturated Fatty Acid Acyl-Coenzyme A Synthetase from the Diatom Thalassiosira pseudonana1

    PubMed Central

    Tonon, Thierry; Qing, Renwei; Harvey, David; Li, Yi; Larson, Tony Robert; Graham, Ian Alexander

    2005-01-01

    The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast. PMID:15821149

  2. Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases: An update

    PubMed Central

    Yan, Sheng; Yang, Xue-Feng; Liu, Hao-Lei; Fu, Nian; Ouyang, Yan; Qing, Kai

    2015-01-01

    Long-chain acyl-CoA synthetase (ACSL) family members include five different ACSL isoforms, each encoded by a separate gene and have multiple spliced variants. ACSLs on endoplasmic reticulum and mitochondrial outer membrance catalyze fatty acids with chain lengths from 12 to 20 carbon atoms to form acyl-CoAs, which are lipid metabolic intermediates and involved in fatty acid metabolism, membrane modifications and various physiological processes. Gain- or loss-of-function studies have shown that the expression of individual ACSL isoforms can alter the distribution and amount of intracellular fatty acids. Changes in the types and amounts of fatty acids, in turn, can alter the expression of intracellular ACSLs. ACSL family members affect not only the proliferation of normal cells, but the proliferation of malignant tumor cells. They also regulate cell apoptosis through different signaling pathways and molecular mechanisms. ACSL members have individual functions in fatty acid metabolism in different types of cells depending on substrate preferences, subcellular location and tissue specificity, thus contributing to liver diseases and metabolic diseases, such as fatty liver disease, obesity, atherosclerosis and diabetes. They are also linked to neurological disorders and other diseases. However, the mechanisms are unclear. This review addresses new findings in the classification and properties of ACSLs and the fatty acid metabolism-associated effects of ACSLs in diseases. PMID:25834313

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

    PubMed Central

    Guo, Fengguang; Ortega-Pierres, Guadalupe; Argüello-García, Raúl; Zhang, Haili; Zhu, Guan

    2015-01-01

    Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids (FA) de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate FA scavenged from the host. ACS is an essential enzyme because FA need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference toward palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis–Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 μM, Ki = 0.18 μM for GiACS1, and IC50 = 2.28 μM, Ki = 0.23 μM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 μM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite. PMID:26257723

  4. Fatty Acid Export from the Chloroplast. Molecular Characterization of a Major Plastidial Acyl-Coenzyme A Synthetase from Arabidopsis1

    PubMed Central

    Schnurr, Judy A.; Shockey, Jay M.; de Boer, Gert-Jan; Browse, John A.

    2002-01-01

    Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS. PMID:12177483

  5. 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

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

    PubMed Central

    Elovson, J

    1975-01-01

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

  7. Hepatic long-chain acyl-CoA synthetase 5 mediates fatty acid channeling between anabolic and catabolic pathways.

    PubMed

    Bu, So Young; Mashek, Douglas G

    2010-11-01

    Long-chain acyl-CoA synthetases (ACSLs) and fatty acid transport proteins (FATPs) activate fatty acids (FAs) to acyl-CoAs prior to their downstream metabolism. Of numerous ACSL and FATP isoforms, ACSL5 is expressed predominantly in tissues with high rates of triacylglycerol (TAG) synthesis, suggesting it may have an anabolic role in lipid metabolism. To characterize the role of ACSL5 in hepatic energy metabolism, we used small interference RNA (siRNA) to knock down ACSL5 in rat primary hepatocytes. Compared with cells transfected with control siRNA, suppression of ACSL5 expression significantly decreased FA-induced lipid droplet formation. These findings were further extended with metabolic labeling studies showing that ACSL5 knockdown resulted in decreased [1-(14)C]oleic acid or acetic acid incorporation into intracellular TAG, phospholipids, and cholesterol esters without altering FA uptake or lipogenic gene expression. ACSL5 knockdown also decreased hepatic TAG secretion proportionate to the observed decrease in neutral lipid synthesis. ACSL5 knockdown did not alter lipid turnover or mediate the effects of insulin on lipid metabolism. Hepatocytes treated with ACSL5 siRNA had increased rates of FA oxidation without changing PPAR-α activity and target gene expression. These results suggest that ACSL5 activates and channels FAs toward anabolic pathways and, therefore, is an important branch point in hepatic FA metabolism. PMID:20798351

  8. Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases.

    PubMed

    Tong, Fumin; Black, Paul N; Coleman, Rosalind A; DiRusso, Concetta C

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation. PMID:16466685

  9. AAE13 encodes a dual-localized malonyl-CoA synthetase that is crucial for mitochondrial fatty acid biosynthesis.

    PubMed

    Guan, Xin; Nikolau, Basil J

    2016-03-01

    Malonyl-CoA is a key intermediate in a number of metabolic processes associated with its role as a substrate in acylation and condensation reactions. These types of reactions occur in plastids, the cytosol and mitochondria, and although carboxylation of acetyl-CoA is the known mechanism for generating the distinct plastidial and cytosolic pools, the metabolic origin of the mitochondrial malonyl-CoA pool is still unclear. In this study we demonstrate that malonyl-CoA synthetase encoded by the Arabidopsis AAE13 (AT3G16170) gene is localized in both the cytosol and the mitochondria. These isoforms are translated from two types of transcripts, one that contains and one that does not contain a mitochondrial-targeting pre-sequence. Whereas the cytosolic AAE13 protein is not essential, due to the presence of a redundant malonyl-CoA generating system provided by a cytosolic acetyl-CoA carboxylase, the mitochondrial AAE13 protein is essential for plant growth. Phenotypes of the aae13-1 mutant are transgenically reversed only if the mitochondrial pre-sequence is present in the ectopically expressed AAE13 proteins. The aae13-1 mutant exhibits typical metabolic phenotypes associated with a deficiency in the mitochondrial fatty acid synthase system, namely depleted lipoylation of the H subunit of the photorespiratory enzyme glycine decarboxylase, increased accumulation of glycine and glycolate and reduced levels of sucrose. Most of these metabolic alterations, and associated morphological changes, are reversed when the aae13-1 mutant is grown in a non-photorespiratory condition (i.e. a 1% CO2 atmosphere), demonstrating that they are a consequence of the deficiency in photorespiration due to the inability to generate lipoic acid from mitochondrially synthesized fatty acids. PMID:26836315

  10. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    SciTech Connect

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

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  11. A novel fatty Acyl-CoA Synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis.

    PubMed

    de Azevedo Souza, Clarice; Kim, Sung Soo; Koch, Stefanie; Kienow, Lucie; Schneider, Katja; McKim, Sarah M; Haughn, George W; Kombrink, Erich; Douglas, Carl J

    2009-02-01

    Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein-ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes. PMID:19218397

  12. Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis*

    PubMed Central

    Witkowski, Andrzej; Thweatt, Jennifer; Smith, Stuart

    2011-01-01

    The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate. PMID:21846720

  13. Endothelial Acyl-CoA Synthetase 1 is not Required for Inflammatory and Apoptotic Effects of a Saturated Fatty Acid-Rich Environment

    PubMed Central

    Li, Xin; Gonzalez, Oscar; Shen, Xia; Barnhart, Shelley; Kramer, Farah; Kanter, Jenny E.; Vivekanandan-Giri, Anuradha; Tsuchiya, Kyoichiro; Handa, Priya; Pennathur, Subramaniam; Kim, Francis; Coleman, Rosalind A.; Schaffer, Jean E.; Bornfeldt, Karin E.

    2013-01-01

    Objective Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells (ECs) and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSL). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating EC responses to a saturated fatty acid-rich environment in vitro and in vivo. Methods and Results Saturated fatty acids caused increased inflammatory activation, ER stress, and apoptosis in mouse microvascular ECs. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and ER stress. However, endothelial ACSL1-deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression. Conclusion Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment. PMID:23241406

  14. Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells.

    PubMed

    Mashek, Douglas G; McKenzie, Michelle A; Van Horn, Cynthia G; Coleman, Rosalind A

    2006-01-13

    Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage. PMID:16263710

  15. Involvement of acyl-CoA synthetase genes in n-alkane assimilation and fatty acid utilization in yeast Yarrowia lipolytica.

    PubMed

    Tenagy; Park, Jun Seok; Iwama, Ryo; Kobayashi, Satoshi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-06-01

    Here, we investigated the roles of YAL1 (FAA1) and FAT1 encoding acyl-CoA synthetases (ACSs) and three additional orthologs of ACS genes FAT2-FAT4 of the yeast Yarrowia lipolytica in the assimilation or utilization of n-alkanes and fatty acids. ACS deletion mutants were generated to characterize their function. The FAT1 deletion mutant exhibited decreased growth on n-alkanes of 10-18 carbons, whereas the FAA1 mutant showed growth reduction on n-alkane of 16 carbons. However, FAT2-FAT4 deletion mutants did not show any growth defects, suggesting that FAT1 and FAA1 are involved in the activation of fatty acids produced during the metabolism of n-alkanes. In contrast, deletions of FAA1 and FAT1-FAT4 conferred no defect in growth on fatty acids. The wild-type strain grew in the presence of cerulenin, an inhibitor of fatty acid synthesis, by utilizing exogenously added fatty acid or fatty acid derived from n-alkane when oleic acid or n-alkane of 18 carbons was supplemented. However, the FAA1 deletion mutant did not grow, indicating a critical role for FAA1 in the utilization of fatty acids. Fluorescent microscopic observation and biochemical analyses suggested that Fat1p is present in the peroxisome and Faa1p is localized in the cytosol and to membranes. PMID:26019148

  16. 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

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

    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

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

    PubMed

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

    2009-01-01

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

  19. 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

  20. Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

    SciTech Connect

    Randhawa, Z.I.; Smith, S.

    1987-03-10

    The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of (/sup 14/C)-labelled peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated M/sub r/ of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.

  1. Distinct transcriptional regulation of long-chain acyl-CoA synthetase isoforms and cytosolic thioesterase 1 in the rodent heart by fatty acids and insulin.

    PubMed

    Durgan, David J; Smith, Justin K; Hotze, Margaret A; Egbejimi, Oluwaseun; Cuthbert, Karalyn D; Zaha, Vlad G; Dyck, Jason R B; Abel, E Dale; Young, Martin E

    2006-06-01

    The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs). Cytosolic thioesterase 1 (CTE1) hydrolyzes cytosolic LCFA-CoAs to LCFAs, generating a potential futile cycle at the expense of ATP utilization. We hypothesized that ACSL isoforms and CTE1 are differentially regulated in the heart during physiological and pathophysiological conditions. Using quantitative RT-PCR, we report that the five known acsl isoforms (acsl1, acsl3, acsl4, acsl5, and acsl6) and cte1 are expressed in whole rat and mouse hearts, as well as adult rat cardiomyocytes (ARCs). Streptozotocin-induced insulin-dependent diabetes (4 wk) and fasting (

  2. Fatty acid induced glioma cell growth is mediated by the acyl-CoA synthetase 5 gene located on chromosome 10q25.1-q25.2, a region frequently deleted in malignant gliomas.

    PubMed

    Yamashita, Y; Kumabe, T; Cho, Y Y; Watanabe, M; Kawagishi, J; Yoshimoto, T; Fujino, T; Kang, M J; Yamamoto, T T

    2000-11-30

    Acyl-CoA synthetase (ACS) ligates fatty acid and CoA to produce acyl-CoA, an essential molecule in fatty acid metabolism and cell proliferation. ACS5 is a recently characterized ACS isozyme highly expressed in proliferating 3T3-L1 cells. Molecular characterization of the human ACS5 gene revealed that the gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683 amino acid protein. Two major ACS5 transcripts of 2.5- and 3.7-kb are distributed in a wide range of tissues with the highest expression in uterus and spleen. Markedly increased levels of ACS5 transcripts were detected in a glioma line, A172 cells, and primary gliomas of grade IV malignancy, while ACS5 expression was found to be low in normal brain. Immunohistochemical analysis also revealed strong immunostaining with an anti-ACS5 antibody in glioblastomas. U87MG glioma cells infected with an adenovirus encoding ACS5 displayed induced cell growth on exposure to palmitate. Consistent with the induction of cell growth, the virus infected cells displayed induced uptake of palmitate. These results demonstrate a novel fatty acid-induced glioma cell growth mediated by ACS5. PMID:11127823

  3. 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. PMID:22299587

  4. Fatty acid-producing hosts

    SciTech Connect

    Pfleger, Brian F; Lennen, Rebecca M

    2013-12-31

    Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

  5. 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 ...

  6. An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

    PubMed Central

    Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

    2014-01-01

    Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids. PMID:25490545

  7. 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.

  8. 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

  9. 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.

  10. Molecular characterization of N-acetylaspartylglutamate synthetase.

    PubMed

    Becker, Ivonne; Lodder, Julia; Gieselmann, Volkmar; Eckhardt, Matthias

    2010-09-17

    The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system. PMID:20643647

  11. 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.

  12. 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 ...

  13. 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 ...

  14. Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

    PubMed

    Beld, Joris; Abbriano, Raffaela; Finzel, Kara; Hildebrand, Mark; Burkart, Michael D

    2016-04-22

    In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short

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

    PubMed

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

    1990-05-01

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

  16. Characterization of the Cephalosporium acremonium pcbAB gene encoding alpha-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains.

    PubMed Central

    Gutiérrez, S; Díez, B; Montenegro, E; Martín, J F

    1991-01-01

    A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha-aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI-BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin-biosynthetic genes. Images PMID:1706706

  17. Impaired expression of acyl-CoA-synthetase 5 in epithelial tumors of the small intestine.

    PubMed

    Gassler, Nikolaus; Schneider, Armin; Kopitz, Jürgen; Schnölzer, Martina; Obermüller, Nicholas; Kartenbeck, Jürgen; Otto, Herwart F; Autschbach, Frank

    2003-10-01

    Fatty acids are implicated in tumorigenesis, but data are limited concerning endogenous fatty acid metabolism of tumor cells in adenomas and adenocarcinomas of the small intestine. The recently cloned human acyl-CoA-synthetase 5 (ACS5) is predominantly found in the small intestine and represents a key enzyme in providing cytosolic acyl-CoA thioesters. Protein synthesis and mRNA expression of ACS5 were studied in human intestinal tissues using different methods, including a newly established monoclonal antibody. In the healthy small intestine, expression of ACS5 was restricted to the villus surface epithelium but was not detectable in enterocytes lining crypts. ACS5 protein and mRNA were progressively diminished in epithelial cells of adenomas and adenocarcinomas of the small intestine. In conclusion, altered expression of ACS5 is probably related to the adenoma-carcinoma sequence of small intestinal epithelial tumors due to an impaired acyl-CoA thioester synthesis. PMID:14608540

  18. Fatty acids - trans fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The data supporting a negative effect of dietary trans fatty acids on cardiovascular disease risk is consistent. The primary dietary sources of trans fatty acids include partially hydrogenated fat and rudiment fat. The adverse effect of trans fatty acids on plasma lipoprotein profiles is consisten...

  19. Aromatase inhibitors and anti-synthetase syndrome.

    PubMed

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

    2016-09-01

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

  20. 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

  1. 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.

  2. Bioluminescent determination of free fatty acids.

    PubMed

    Kather, H; Wieland, E

    1984-08-01

    A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes. PMID:6486422

  3. 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

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

    PubMed Central

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

    2015-01-01

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

  5. 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

  6. The microsomal dicarboxylyl-CoA synthetase.

    PubMed Central

    Vamecq, J; de Hoffmann, E; Van Hoof, F

    1985-01-01

    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo. PMID:4062873

  7. 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

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

    PubMed

    Wang, Yechun; Chen, Hui; Yu, Oliver

    2014-06-01

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

  9. 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

  10. 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

  11. Site-specific cleavage of acetoacetyl-CoA synthetase by legumain.

    PubMed

    Hasegawa, Shinya; Inoue, Daiki; Yamasaki, Masahiro; Li, Chuan; Imai, Masahiko; Takahashi, Noriko; Fukui, Tetsuya

    2016-06-01

    Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme and is responsible for the synthesis of cholesterol and fatty acids. We have previously shown that AACS is cleaved by legumain, a lysosomal asparaginyl endopeptidase. In this study, we attempted to determine the cleavage site of AACS. Mutagenesis analysis of AACS revealed that Asn547 is the specific cleavage site of AACS in mouse livers. The cleaved form of AACS (1-547) lost the ability to convert acetoacetate to acetoacetyl-CoA. Moreover, hydrodynamics-based gene transduction showed that overexpression of AACS (1-547) increases the protein expression of caveolin-1, the principal component of the caveolae. These results suggest that cleavage of AACS by legumain is critical for the regulation of enzymatic activity and results in gain-of-function changes. PMID:27129883

  12. 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 ...

  13. Continuous spectrophotometric assay for aminoacyl-tRNA synthetases.

    PubMed

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

    1984-11-01

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

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

    PubMed

    Fox, Barbara A; Bzik, David J

    2003-01-01

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

  15. 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.

  16. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    PubMed Central

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  17. Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis.

    PubMed

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

    2010-12-01

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

  18. 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. PMID:25437123

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

    SciTech Connect

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

    1988-12-13

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

  20. Regulation of Glutamine Synthetase V. Partial Purification and Properties of Glutamine Synthetase from Bacillus licheniformis

    PubMed Central

    Hubbard, Jerry S.; Stadtman, E. R.

    1967-01-01

    The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn++ was required for activity, and the requirement cannot be satisfied with Mg++. Maximal activity of the biosynthetic reaction was observed when ATP and Mn++ were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn++. A complex of ATP and Mn++ is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active. PMID:6051339

  1. Biosynthesis of medium-chain fatty acids by mammary epithelial cells from virgin rats.

    PubMed Central

    Smith, S; Pasco, D; Nandi, S

    1983-01-01

    Epithelial cells were isolated from the undifferentiated mammary glands of mature virgin female rats, and their lipogenic characteristics were studied. These cells synthesized predominantly medium-chain fatty acids, albeit at a low rate. In contrast, whole tissue from mammary glands of virgin rats synthesized predominantly long-chain fatty acids at a relatively higher rate, indicating that the lipogenic activity is dominated by the adipocyte component of the gland. Enzyme assays revealed that thioesterase II, the enzyme which regulates production of medium-chain fatty acids by the fatty acid synthetase, was present at a high activity in the undifferentiated mammary epithelial cells of virgin rats. Immunohistochemical studies confirmed this observation and showed that the regulatory enzyme was present exclusively in the epithelial cells lining the alveolar and ductal elements of the undifferentiated gland. This study demonstrates that the potential to elaborate tissue-specific medium-chain fatty acids is already expressed in the undifferentiated tissue of virgin rats and is not acquired as a result of the differentiation associated with the lactogenic phase of development. In this species mammary epithelial cells apparently synthesize predominantly medium-chain fatty acids at all stages of development, and only the overall rate of synthesis is increased on induction of the fatty acid synthetase during lactogenesis. Images Fig. 1. Fig. 2. PMID:6409098

  2. Long-chain polyunsaturated fatty acids stimulate cellular fatty acid uptake in human placental choriocarcinoma (BeWo) cells.

    PubMed

    Johnsen, G M; Weedon-Fekjaer, M S; Tobin, K A R; Staff, A C; Duttaroy, A K

    2009-12-01

    Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 microM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [(14)C] fatty acids was increased by 20-50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ASCL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development. PMID:19880178

  3. 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

  4. [Thromboxane A2 synthetase inhibitor in asthma therapy].

    PubMed

    Machida, K; Takagi, K; Horiba, M

    1996-11-01

    Thromboxane A2(TXA2), a platelet aggregator and vasoconstricter, has been implicated as a potential mediator of bronchial asthma. TXA2 induces potent contraction of airway smooth muscles and airway hyperresponsiveness. OKY-046 (ozagrel hydrochloride) is a specific inhibitor of TXA2 synthetase and a new antiasthmatic agent. In a phase III study ozagrel has shown significantly higher effect in ameliorating the asthma symptoms and reduced the dose of concomitant steroid therapy compared to azelastine hydrochloride. Both basical and clinical studies showed that TXA2 synthetase inhibitor is effective on airway hyperresponsiveness. In this review the role of TXA2 synthetase inhibitor in current asthma therapy, which is based on the Japanese guideline of allergic disorders, was discussed. PMID:8950950

  5. tRNA synthetase: tRNA Aminoacylation and beyond

    PubMed Central

    Pang, Yan Ling Joy; Poruri, Kiranmai; Martinis, Susan A.

    2014-01-01

    The aminoacyl-tRNA synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. Each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of tRNA isoacceptors. These ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their aminoacylation role and impact a wide range of other metabolic pathways and cell signaling processes. The family of aminoacyl-tRNA synthetases have also been suggested as a remarkable scaffold to incorporate new domains that would drive evolution and the emergence of new organisms with more complex function. Because they are essential, the tRNA synthetases have served as pharmaceutical targets for drug and antibiotic development. The recent unfolding of novel important functions for this family of proteins offers new and promising pathways for therapeutic development to treat diverse human diseases. PMID:24706556

  6. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

    PubMed

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

    2010-12-01

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

  7. 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

  8. Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes.

    PubMed

    Ueda, H; Yoshihara, Y; Fukushima, N; Shiomi, H; Nakamura, A; Takagi, H

    1987-06-15

    Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation. PMID:3597366

  9. Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis.

    PubMed

    Reinartz, Andrea; Ehling, Josef; Leue, Andrea; Liedtke, Christian; Schneider, Ursula; Kopitz, Jürgen; Weiss, Thomas; Hellerbrand, Claus; Weiskirchen, Ralf; Knüchel, Ruth; Gassler, Nikolaus

    2010-09-01

    In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFalpha-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders. PMID:20470896

  10. 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.

  11. 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

  12. 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. PMID:25385624

  13. 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

  14. N-acetylaspartylglutamate synthetase II synthesizes N-acetylaspartylglutamylglutamate.

    PubMed

    Lodder-Gadaczek, Julia; Becker, Ivonne; Gieselmann, Volkmar; Wang-Eckhardt, Lihua; Eckhardt, Matthias

    2011-05-13

    N-Acetylaspartylglutamate (NAAG) is found at high concentrations in the vertebrate nervous system. NAAG is an agonist at group II metabotropic glutamate receptors. In addition to its role as a neuropeptide, a number of functions have been proposed for NAAG, including a role as a non-excitotoxic transport form of glutamate and a molecular water pump. We recently identified a NAAG synthetase (now renamed NAAG synthetase I, NAAGS-I), encoded by the ribosomal modification protein rimK-like family member B (Rimklb) gene, as a member of the ATP-grasp protein family. We show here that a structurally related protein, encoded by the ribosomal modification protein rimK-like family member A (Rimkla) gene, is another NAAG synthetase (NAAGS-II), which in addition, synthesizes the N-acetylated tripeptide N-acetylaspartylglutamylglutamate (NAAG(2)). In contrast, NAAG(2) synthetase activity was undetectable in cells expressing NAAGS-I. Furthermore, we demonstrate by mass spectrometry the presence of NAAG(2) in murine brain tissue and sciatic nerves. The highest concentrations of both, NAAG(2) and NAAG, were found in sciatic nerves, spinal cord, and the brain stem, in accordance with the expression level of NAAGS-II. To our knowledge the presence of NAAG(2) in the vertebrate nervous system has not been described before. The physiological role of NAAG(2), e.g. whether it acts as a neurotransmitter, remains to be determined. PMID:21454531

  15. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  16. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    PubMed Central

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts. PMID:10639127

  17. 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.

  18. Sporopollenin Biosynthetic Enzymes Interact and Constitute a Metabolon Localized to the Endoplasmic Reticulum of Tapetum Cells[W

    PubMed Central

    Lallemand, Benjamin; Erhardt, Mathieu; Heitz, Thierry; Legrand, Michel

    2013-01-01

    The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon. PMID:23632852

  19. Sporopollenin biosynthetic enzymes interact and constitute a metabolon localized to the endoplasmic reticulum of tapetum cells.

    PubMed

    Lallemand, Benjamin; Erhardt, Mathieu; Heitz, Thierry; Legrand, Michel

    2013-06-01

    The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon. PMID:23632852

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

    PubMed

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

    2014-04-16

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

  1. Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism.

    PubMed

    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

  2. Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse)

    PubMed Central

    Takahashi, Tetsuyuki; Nishida, Takeshi; Baba, Hayato; Hatta, Hideki; Imura, Johji; Sutoh, Mitsuko; Toyohara, Syunji; Hokao, Ryoji; Watanabe, Syunsuke; Ogawa, Hirohisa; Uehara, Hisanori; Tsuneyama, Koichi

    2016-01-01

    We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of β-catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence. PMID:27446562

  3. A Peroxisomal Long-Chain Acyl-CoA Synthetase from Glycine max Involved in Lipid Degradation

    PubMed Central

    Jiang, Bingjun; Sun, Xuegang; Gu, Shoulai; Han, Tianfu; Hou, Wensheng

    2014-01-01

    Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination. PMID:24992019

  4. 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

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

    PubMed

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

    2015-01-12

    A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment. PMID:25584894

  6. Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism.

    PubMed

    Chen, Liwei; Zhang, Jianhua; Lee, Jaslyn; Chen, Wei Ning

    2014-08-01

    Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels. PMID:24769906

  7. Bioactive Fatty Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxygenated fatty acids are useful as specialty chemicals, plasticizers, and biomedicals. Microbial enzymes convert fatty acids to mono-, di-, and trihydroxy fatty acid products. Among them, Bacillus megaterium ALA2 converted n-6 and n-3 PUFAs to many new oxygenated fatty acids. Linoleic acid was ...

  8. Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

    PubMed Central

    Ravishankar, Sudha; Ambady, Anisha; Swetha, Rayapadi G.; Anbarasu, Anand; Ramaiah, Sudha; Sambandamurthy, Vasan K.

    2016-01-01

    Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis. PMID:26794499

  9. Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

    PubMed Central

    Priefert, H; Steinbüchel, A

    1992-01-01

    The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54. Images PMID:1356967

  10. Plant fatty acid hydroxylases

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  11. Fat Metabolism in Higher Plants XXXVI: Long Chain Fatty Acid Synthesis in Germinating Peas 1

    PubMed Central

    Macey, Michael J. K.; Stumpf, P. K.

    1968-01-01

    A low lipid, high starch containing tissue, namely cotyledons of germinating pea seedlings was examined for its capacity to synthesize fatty acid. Intact tissue slices readily incorporate acetate-14C into fatty acids from C16 to C24. Although crude homogenates synthesize primarily 16:0 and 18:0 from malonyl CoA, subsequent fractionation into a 10,000g pellet, a 105g pellet and supernatant (soluble synthetase) revealed that the 105g pellet readily synthesizes C16 to C28 fatty acids whereas the 10,000g and the supernatant synthesize primarily C16 and C18. All systems require acyl carrier protein (ACP), TPNH, DPNH if malonyl CoA is the substrate and ACP, Mg2+, CO2, ATP, TPNH, and DPNH if acetyl CoA is the substrate. The cotyledons of germinating pea seedlings appear to have a soluble synthetase and 10,000g particles for the synthesis of C16 and C18 fatty acid, and 105g particles which specifically synthesize the very long chain fatty acid from malonyl CoA, presumably via malonyl ACP. PMID:16656949

  12. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  13. Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Napier, Johnathan A.; Chye, Mee-Len

    2014-01-01

    Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis. PMID:25264899

  14. 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

  15. Glutamine synthetase gene evolution: a good molecular clock.

    PubMed Central

    Pesole, G; Bozzetti, M P; Lanave, C; Preparata, G; Saccone, C

    1991-01-01

    Glutamine synthetase (EC 6.3.1.2) gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. Our calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. Our data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves. PMID:1671172

  16. 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

  17. Promotion of glioma cell survival by acyl-CoA synthetase 5 under extracellular acidosis conditions.

    PubMed

    Mashima, T; Sato, S; Sugimoto, Y; Tsuruo, T; Seimiya, H

    2009-01-01

    Extracellular acidosis (low pH) is a tumor microenvironmental stressor that has a critical function in the malignant progression and metastatic dissemination of tumors. To survive under stress conditions, tumor cells must evolve resistance to stress-induced toxicity. Acyl-CoA synthetase 5 (ACSL5) is a member of the ACS family, which converts fatty acid to acyl-CoA. ACSL5 is frequently overexpressed in malignant glioma, whereas its functional significance is still unknown. Using retrovirus-mediated stable gene transfer (gain of function) and small interfering RNA-mediated gene silencing (loss of function), we show here that ACSL5 selectively promotes human glioma cell survival under extracellular acidosis. ACSL5 enhanced cell survival through its ACS catalytic activity. To clarify the genome-wide changes in cell signaling pathways by ACSL5, we performed cDNA microarray analysis and identified an ACSL5-dependent gene expression signature. The analysis revealed that ACSL5 was critical to the expression of tumor-related factors including midkine (MDK), a heparin-binding growth factor frequently overexpressed in cancer. Knockdown of MDK expression significantly attenuated ACSL5-mediated survival under acidic state. These results indicate that ACSL5 is a critical factor for survival of glioma cells under acidic tumor microenvironment, thus providing novel molecular basis for cancer therapy. PMID:18806831

  18. Mouse very long-chain acyl-CoA synthetase in X-linked adrenoleukodystrophy.

    PubMed

    Heinzer, Ann K; Kemp, Stephan; Lu, Jyh-Feng; Watkins, Paul A; Smith, Kirby D

    2002-08-01

    X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by accumulation of very long-chain fatty acids (VLCFA). This accumulation has been attributed to decreased VLCFA beta-oxidation and peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity. The X-ALD gene, ABCD1, encodes a peroxisomal membrane ATP binding cassette transporter, ALDP, that is hypothesized to affect VLCS activity in peroxisomes by direct interaction with the VLCS enzyme. Recently, a VLCS gene that encodes a protein with significant sequence identity to known rat and human peroxisomal VLCS protein has been identified in mice. We find that the mouse VLCS gene (Vlcs) encodes an enzyme (Vlcs) with VLCS activity that localizes to peroxisomes and is expressed in X-ALD target tissues. We show that the expression of Vlcs in the peroxisomes of X-ALD mouse fibroblasts improves VLCFA beta-oxidation in these cells, implying a role for this enzyme in the biochemical abnormality of X-ALD. X-ALD mice, which accumulate VLCFA in tissues, show no change in the expression of Vlcs, the subcellular localization of Vlcs, or general peroxisomal VLCS activity. These observations imply that ALDP is not necessary for the proper expression or localization of Vlcs protein, and the control of VLCFA levels does not depend on the direct interaction of Vlcs and ALDP. PMID:12048192

  19. Explorations of Catalytic Domains in Non-Ribosomal Peptide Synthetase Enzymology

    PubMed Central

    Hur, Gene H.; Vickery, Christopher R.; Burkart, Michael D.

    2016-01-01

    Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Originating from bacteria and fungi, these peptide-based natural products consist not only of the 20 canonical L-amino acids, but also non-proteinogenic amino acids, heterocyclic rings, sugars, and fatty acids, generating tremendous chemical diversity. As a result, these secondary metabolites exhibit a broad array of bioactivity ranging from antimicrobial to anticancer. The biosynthesis of these complex compounds is carried out by large multimodular megaenzymes called nonribosomal peptide synthetases (NRPSs). Each module is responsible for incorporation of a monomeric unit into the natural product peptide and is composed of individual domains that perform different catalytic reactions. Biochemical and bioinformatic investigations of these enzymes have uncovered the key principles of NRP synthesis, expanding the pharmaceutical potential of their enzymatic processes. Progress has been made in the manipulation of this biosynthetic machinery to develop new chemoenzymatic approaches for synthesizing novel pharmaceutical agents with increased potency. This review focuses on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the discrete domains within NRPSs. PMID:22802156

  20. Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase.

    PubMed Central

    Foor, F; Cedergren, R J; Streicher, S L; Rhee, S G; Magasanik, B

    1978-01-01

    The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA. PMID:26659

  1. Beta-oxidation of very-long-chain fatty acids and their coenzyme A derivatives by human skin fibroblasts.

    PubMed

    Singh, H; Derwas, N; Poulos, A

    1987-05-01

    The beta-oxidation of lignoceric acid (C24:0), hexacosanoic acid (C26:0), and their coenzyme A derivatives was investigated in human skin fibroblast homogenates. The cofactor requirements for oxidation of lignoceric acid and hexacosanoic acid were identical but were different from their coenzyme A derivatives. For example, lignoceric acid and hexacosanoic acid oxidation was strictly ATP dependent whereas the oxidation of the corresponding coenzyme A derivatives was ATP independent. Also the rate of oxidation of coenzyme A derivatives of lignoceric acid or hexacosanoic acid was much higher compared to the free fatty acids. In patients with Zellweger's syndrome, X-linked adrenoleukodystrophy and infantile Refsum's disease, the beta-oxidation of lignoceric and hexacosanoic acids was defective whereas the oxidation of their corresponding coenzyme A derivatives was nearly normal. The results presented in this communication suggest strongly that the beta-oxidation of very-long-chain fatty acids occurs exclusively in peroxisomes. However, the coenzyme A derivatives of very-long-chain fatty acids can be oxidized in mitochondria as well as in peroxisomes. The inability of the mitochondrial system to oxidize free fatty acids may be due to its inability to convert them to their corresponding coenzyme A derivatives. Our results suggest that a specific very-long-chain fatty acyl CoA synthetase may be required for the activation of the free fatty acids and that this synthetase may be deficient in patients with Zellweger's syndrome and possibly X-linked adrenoleukodystrophy, as well. The results presented suggest that substrate specificity and the subcellular localization of the synthetase may regulate the beta-oxidation of very-long-chain fatty acids in the cell. PMID:2437859

  2. 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.

  3. 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

  4. Omega-3 Fatty Acids

    MedlinePlus

    Omega-3 fatty acids are used together with lifestyle changes (diet, weight-loss, exercise) to reduce the amount ... the blood in people with very high triglycerides. Omega-3 fatty acids are in a class of medications ...

  5. Omega-6 Fatty Acids

    MedlinePlus

    Omega-6 fatty acids are types of fats. Some types are found in vegetable oils, including corn, evening primrose seed, safflower, and soybean oils. Other types of omega-6 fatty acids are found in black currant seed, borage seed, ...

  6. Omega-6 Fatty Acids

    MedlinePlus

    ... types of fats. Some types are found in vegetable oils, including corn, evening primrose seed, safflower, and soybean ... from studying specific omega-6 fatty acids or plant oils containing omega-6 fatty acids. See the separate ...

  7. Fatty acid analogs

    DOEpatents

    Elmaleh, David R.; Livni, Eli

    1985-01-01

    In one aspect, a radioactively labeled analog of a fatty acid which is capable of being taken up by mammalian tissue and which exhibits an in vivo beta-oxidation rate below that with a corresponding radioactively labeled fatty acid.

  8. Production of free monounsaturated fatty acids by metabolically engineered Escherichia coli

    PubMed Central

    2014-01-01

    Background Monounsaturated fatty acids (MUFAs) are the best components for biodiesel when considering the low temperature fluidity and oxidative stability. However, biodiesel derived from vegetable oils or microbial lipids always consists of significant amounts of polyunsaturated and saturated fatty acids (SFAs) alkyl esters, which hampers its practical applications. Therefore, the fatty acid composition should be modified to increase MUFA contents as well as enhancing oil and lipid production. Results The model microorganism Escherichia coli was engineered to produce free MUFAs. The fatty acyl-ACP thioesterase (AtFatA) and fatty acid desaturase (SSI2) from Arabidopsis thaliana were heterologously expressed in E. coli BL21 star(DE3) to specifically release free unsaturated fatty acids (UFAs) and convert SFAs to UFAs. In addition, the endogenous fadD gene (encoding acyl-CoA synthetase) was disrupted to block fatty acid catabolism while the native acetyl-CoA carboxylase (ACCase) was overexpressed to increase the malonyl coenzyme A (malonyl-CoA) pool and boost fatty acid biosynthesis. The finally engineered strain BL21ΔfadD/pE-AtFatAssi2&pA-acc produced 82.6 mg/L free fatty acids (FFAs) under shake-flask conditions and FFAs yield on glucose reached about 3.3% of the theoretical yield. Two types of MUFAs, palmitoleate (16:1Δ9) and cis-vaccenate (18:1Δ11) made up more than 75% of the FFA profiles. Fed-batch fermentation of this strain further enhanced FFAs production to a titer of 1.27 g/L without affecting fatty acid compositions. Conclusions This study demonstrated the possibility to regulate fatty acid composition by using metabolic engineering approaches. FFAs produced by the recombinant E. coli strain consisted of high-level MUFAs and biodiesel manufactured from these fatty acids would be more suitable for current diesel engines. PMID:24716602

  9. Assignment of the cysteinyl-tRNA synthetase gene (CARS) to 11p15. 5

    SciTech Connect

    Cruzen, M.E.; Bengtsson, U.; McMahon, J.; Wasmuth, J.J.; Arfin, S.M. )

    1993-03-01

    The attachment of each of the 20 naturally occurring amino acids to their cognate tRNA isoaccepting families is catalyzed by a specific aminoacyl-tRNA synthetase. The structural genes encoding 10 of these enzymes have been assigned to specific human chromosomes. The HARS, LARS, RARS, and TARS genes, encoding histidyl-, leucyl-, arginyl-, and threonyl-tRNA synthetases, respectively, are all located on chromosome 5( 1, 5, 7, 9, 14). The MARS (methionyl-tRNA synthetase), NARS (asparaginyl-tRNA synthetase), VARS (valyl-tRNA synthetase), and WARS (tryptophanyl-tRNA synthetase) genes have been assigned to chromosomes 12, 18, 6, and 14, respectively (3, 4, 6, 8). A gene originally identified as encoding glutaminyl-tRNA synthetase was mapped to chromosome 1q32-q42 (10). However, a recent study suggests that the product of this gene is, in fact, a multifunctional enzyme with both glutamyl- and prolyl-tRNA synthetase activities (2). The fact that 4 of the 10 aminoacyl-tRNA synthetase genes already mapped are located on chromosome 5 may be fortuitous but might also indicate an evolutionary or regulatory relatedness. It is therefore, of interest to map genes encoding other aminoacyl-tRNA synthetases to determine if additional examples of synteny exist. The recent isolation of cDNA and genomic DNA clones for human cysteinyl-tRNA synthetase has now enabled us to map the CARS gene to segment p15.5 on chromosome 11 by fluorescence in situ hybridization.

  10. Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi

    PubMed Central

    Ibba, Michael; Bono, James L.; Rosa, Patricia A.; Söll, Dieter

    1997-01-01

    Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics. PMID:9405621

  11. 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

  12. 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

  13. 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

  14. DOES IRON OR HEME CONTROL RAT HEPATIC DELTA-AMINOLEVULINIC ACID SYNTHETASE ACTIVITY

    EPA Science Inventory

    Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic d-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter d-aminolevulinic acid synthetase activity and ha...

  15. 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.

  16. 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.

  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, 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.

  18. 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.

  19. Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

    PubMed Central

    Kozloff, L. M.; Lute, M.; Crosby, L. K.

    1977-01-01

    Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td+, whereas particles produced after infection with td− had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from tdts to td+ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both tdts and frdts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frdts mutants to frd+ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles. PMID:894793

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

    PubMed

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

    1992-10-01

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

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

    PubMed Central

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

    1992-01-01

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

  2. 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

  3. 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

  4. Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids.

    PubMed

    Metz, James G; Kuner, Jerry; Rosenzweig, Bradley; Lippmeier, James C; Roessler, Paul; Zirkle, Ross

    2009-06-01

    In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg(2+) to in vitro assays facilitates appearance of radiolabel from (14)C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase. PMID:19272783

  5. Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver.

    PubMed

    Cabrero, C; Puerta, J; Alemany, S

    1987-12-30

    Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction. PMID:3121322

  6. Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

    PubMed Central

    Nemoto, Yoshihisa; Toda, Katsumi; Ono, Masafumi; Fujikawa-Adachi, Kiyomi; Saibara, Toshiji; Onishi, Saburo; Enzan, Hideaki; Okada, Teruhiko; Shizuta, Yutaka

    2000-01-01

    Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid β-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid β-oxidation. In vitro assays of fatty acid β-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid β-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17β-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid β-oxidation and in maintaining hepatic lipid homeostasis. PMID:10862797

  7. Very Long-Chain Acyl-CoA Synthetase 3: Overexpression and Growth Dependence in Lung Cancer

    PubMed Central

    Pei, Zhengtong; Fraisl, Peter; Shi, Xiaohai; Gabrielson, Edward; Forss-Petter, Sonja; Berger, Johannes; Watkins, Paul A.

    2013-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65–76%, and the ability of tumor cells to form colonies in soft agar suspension by 65–80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is

  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. Management of a patient with holocarboxylase synthetase deficiency.

    PubMed

    Van Hove, Johan L K; Josefsberg, Sagi; Freehauf, Cynthia; Thomas, Janet A; Thuy, Le Phuc; Barshop, Bruce A; Woontner, Michael; Mock, Donald M; Chiang, Pei-Wen; Spector, Elaine; Meneses-Morales, Iván; Cervantes-Roldán, Rafael; León-Del-Río, Alfonso

    2008-12-01

    We investigated in a patient with holocarboxylase synthetase deficiency, the relation between the biochemical and genetic factors of the mutant protein with the pharmacokinetic factors of successful biotin treatment. A girl exhibited abnormal skin at birth, and developed in the first days of life neonatal respiratory distress syndrome and metabolic abnormalities diagnostic of multiple carboxylase deficiency. Enzyme assays showed low carboxylase activities. Fibroblast analysis showed poor incorporation of biotin into the carboxylases, and low transfer of biotin by the holocarboxylase synthetase enzyme. Kinetic studies identified an increased Km but a preserved Vmax. Mutation analysis showed the child to be a compound heterozygote for a new nonsense mutation Q379X and for a novel missense mutation Y663H. This mutation affects a conserved amino acid, which is located the most 3' of all recorded missense mutations thus far described, and extends the region of functional biotin interaction. Treatment with biotin 100mg/day gradually improved the biochemical abnormalities in blood and in cerebrospinal fluid (CSF), corrected the carboxylase enzyme activities, and provided clinical stability and a normal neurodevelopmental outcome. Plasma concentrations of biotin were increased to more than 500 nM, thus exceeding the increased Km of the mutant enzyme. At these pharmacological concentrations, the CSF biotin concentration was half the concentration in blood. Measuring these pharmacokinetic variables can aid in optimizing treatment, as individual tailoring of dosing to the needs of the mutation may be required. PMID:18974016

  10. MANAGEMENT OF A PATIENT WITH HOLOCARBOXYLASE SYNTHETASE DEFICIENCY

    PubMed Central

    Van Hove, Johan LK; Josefsberg, Sagi; Freehauf, Cynthia; Thomas, Janet A.; Thuy, Le Phuc; Barshop, Bruce A.; Woontner, Michael; Mock, Donald M; Chiang, Pei-Wen; Spector, Elaine; Meneses-Morales, Iván; Cervantes-Roldán, Rafael; León-Del-Río, Alfonso

    2009-01-01

    We investigated in a patient with holocarboxylase synthetase deficiency, the relation between the biochemical and genetic factors of the mutant protein with the pharmacokinetic factors of successful biotin treatment. A girl exhibited abnormal skin at birth, and developed in the first days of life neonatal respiratory distress syndrome and metabolic abnormalities diagnostic of multiple carboxylase deficiency. Enzyme assays showed low carboxylase activities. Fibroblast analysis showed poor incorporation of biotin into the carboxylases, and low transfer of biotin by the holocarboxylase synthetase enzyme. Kinetic studies identified an increased Km but a preserved Vmax. Mutation analysis showed the child to be a compound heterozygote for a new nonsense mutation Q379X and for a novel missense mutation Y663H. This mutation affects a conserved amino acid, which is located the most 3′ of all recorded missense mutations thus far described, and extends the region of functional biotin interaction. Treatment with biotin 100 mg/day gradually improved the biochemical abnormalities in blood and in cerebrospinal fluid, corrected the carboxylase enzyme activities, and provided clinical stability and a normal neurodevelopmental outcome. Plasma concentrations of biotin were increased to more than 500 nM, thus exceeding the increased Km of the mutant enzyme. At these pharmacological concentrations, the CSF biotin concentration was half the concentration in blood. Measuring these pharmacokinetic variables can aid in optimizing treatment, as individual tailoring of dosing to the needs of the mutation may be required. PMID:18974016

  11. Novel Insights into Regulation of Asparagine Synthetase in Conifers

    PubMed Central

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

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed. PMID:22654888

  12. Holocarboxylase synthetase deficiency pre and post newborn screening

    PubMed Central

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

    2016-01-01

    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

  13. 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

  14. Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

    PubMed

    Denis, Laurence; Grossemy, Marie; Douce, Roland; Alban, Claude

    2002-03-22

    Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation. PMID:11784724

  15. N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

    PubMed Central

    Hassan, Yousef I.; Moriyama, Hideaki; Olsen, Lars J.; Bi, Xin; Zempleni, Janos

    2009-01-01

    Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS. PMID:19157941

  16. Direct evidence for an acyl phosphate intermediate in the folylpoly-. gamma. -glutamate synthetase and dihydrofolate synthetase-catalyzed reactions

    SciTech Connect

    Banerjee, R.

    1987-01-01

    The mechanism of the reactions catalyzed by two enzymes, namely dihydrofolate synthetase (DHFS) and folylpoly-..gamma..-glutamate synthetase (FPGS), has been investigated. The nature of the intermediate in each of the two reactions was monitored simultaneously in the multifunctional enzyme, FPGS/DHFS from E. coli. The latter was isolated from a transformant containing the cloned FPGS/DHFS gene. Incubation of (/sup 18/O)-H/sub 2/Pte and (/sup 17/O)-glutamate with ATP and the enzyme, resulted in the formation of (/sup 18/O)- and (/sup 17/O)-P/sub i/, thus providing strong evidence for the formation of an acyl phosphate species during catalysis of each reaction. The inorganic phosphate formed in the enzyme-catalyzed reaction was purified by chromatography on DEAE-cellulose, then converted to the trimethyl ester and analyzed by mass spectroscopy /sup 17/O NMR and /sup 31/P NMR. Stoichiometric formation of (/sup 17/O)- and (/sup 18/O)-Pi was observed. /sup 31/P NMR analysis showed the expected /sup 18/O-induced isotopic perturbations. The presence of (/sup 17/O)-trimethyl phosphate was revealed by /sup 17/O NMR. The mechanism of the FPGS-catalyzed reaction was also investigated with the antifolate (/sup 18/O)-methotrexate.

  17. Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites☆

    PubMed Central

    Pham, James S.; Dawson, Karen L.; Jackson, Katherine E.; Lim, Erin E.; Pasaje, Charisse Flerida A.; Turner, Kelsey E.C.; Ralph, Stuart A.

    2013-01-01

    Aminoacyl-tRNA synthetases are central enzymes in protein translation, providing the charged tRNAs needed for appropriate construction of peptide chains. These enzymes have long been pursued as drug targets in bacteria and fungi, but the past decade has seen considerable research on aminoacyl-tRNA synthetases in eukaryotic parasites. Existing inhibitors of bacterial tRNA synthetases have been adapted for parasite use, novel inhibitors have been developed against parasite enzymes, and tRNA synthetases have been identified as the targets for compounds in use or development as antiparasitic drugs. Crystal structures have now been solved for many parasite tRNA synthetases, and opportunities for selective inhibition are becoming apparent. For different biological reasons, tRNA synthetases appear to be promising drug targets against parasites as diverse as Plasmodium (causative agent of malaria), Brugia (causative agent of lymphatic filariasis), and Trypanosoma (causative agents of Chagas disease and human African trypanosomiasis). Here we review recent developments in drug discovery and target characterisation for parasite aminoacyl-tRNA synthetases. PMID:24596663

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

    PubMed

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

    2008-12-01

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

  19. Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases.

    PubMed Central

    Cusack, S; Härtlein, M; Leberman, R

    1991-01-01

    Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain. Images PMID:1852601

  20. Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

    PubMed Central

    Ray, Peter M.

    1973-01-01

    Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent β-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 μm IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control. PMID:16658379

  1. Properties of Kaurene Synthetase from Marah macrocarpus1

    PubMed Central

    Frost, Russell G.; West, Charles A.

    1977-01-01

    The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000). The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg2+ > Mn2+ > Co2+. Activities A and B were both sensitive to inhibition by Hg2+, Cu2+, p-hydroxymercuribenzoate, and N-ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km′ (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 μm. Values of 0.5 and 0.6 μm were obtained for Km′ and Km, respectively, for copalyl-pyrophosphate. The Vm′ values for the two activities were similar: 12 and 9 pmol/minute·μg protein for activities A and B, respectively. N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4-dichlorobenzylammonium chloride (Phosfon S), 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N-heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 μm, were effective inhibitors of kaurene

  2. 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

  3. 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

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

    PubMed

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

    1990-12-01

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

  5. Multiple molecular forms of glutamine synthetase in pea seeds.

    PubMed

    Antonyuk, L P; Pushkin, A V; Vorobyeva, L M; Solovjeva, N A; Evstigneeva, Z G; Kretovich, W L

    1982-08-20

    Multiple molecular forms of glutamine synthetase (GS, EC 6.3.1.2) have been studied in pea seeds of different varieties. The number of GS molecular forms in the seeds proved to be related to their colour. Two GS forms in the green seeds have been found and only one of them in the yellow seeds. Green seeds had chlorophyll content amounted to 0.4% of the total pigment content in the leaves. Chloroplasts, somewhat smaller than those in pea leaves of the same variety, have been isolated from green seeds. The presence of the second GS form in the pea green seeds we relate to the chloroplasts. By electrophoretic mobility both forms of GS from the green seeds are not identical to the chloroplast GS and the cytosol GS of leaves. Thus, we believe pea plant to contain, at least, four GS forms. PMID:6127624

  6. Aminoacyl-tRNA synthetase complexes: molecular multitasking revealed

    PubMed Central

    Hausmann, Corinne D.; Ibba, Michael

    2008-01-01

    The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. An overview of the complexes aaRSs form in all domains of life is presented, demonstrating the extensive network of connections between the translational machinery and cellular components involved in a myriad of essential processes beyond protein synthesis. PMID:18522650

  7. S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

    PubMed

    Yarlett, N; Garofalo, J; Goldberg, B; Ciminelli, M A; Ruggiero, V; Sufrin, J R; Bacchi, C J

    1993-03-24

    S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism. PMID:8457607

  8. 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)

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

    PubMed

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

    2006-09-26

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

  10. Fats and fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The absolute fat requirement of the human species is the amount of essential fatty acids needed to maintain optimal fatty acid composition of all tissues and normal eicosanoid synthesis. At most, this requirement is no more than about 5% of an adequate energy intake. However, fat accounts for appro...

  11. Omega-3 Fatty Acids

    MedlinePlus

    Omega-3 fatty acids are used together with lifestyle changes (diet, weight-loss, exercise) to reduce the amount of triglycerides (a fat-like ... people with very high triglycerides. Omega-3 fatty acids are in a class of medications called antilipemic ...

  12. Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium

    SciTech Connect

    Wada, M.; Fukunaga, N.; Sasaki, S. )

    1989-08-01

    Biosynthesis of palmitic, palmitoleic, and cis-vaccenic acids in Pseudomonas sp. strain E-3 was investigated with in vitro and in vivo systems. (1-{sup 14}C)palmitic acid was aerobically converted to palmitoleate and cis-vaccenate, and the radioactivities on their carboxyl carbons were 100 and 43%, respectively, of the total radioactivity in the fatty acids. Palmitoyl coenzyme A desaturase activity was found in the membrane fraction. (1-{sup 14}C)stearic acid was converted to octadecenoate and C16 fatty acids. The octadecenoate contained oleate and cis-vaccenate, but only oleate was produced in the presence of cerulenin. (1-{sup 14}C)lauric acid was aerobically converted to palmitate, palmitoleate, and cis-vaccenate. Under anaerobic conditions, palmitate (62%), palmitoleate (4%), and cis-vaccenate (34%) were produced from (1-{sup 14}C)acetic acid, while they amounted to 48, 39, and 14%, respectively, under aerobic conditions. In these incorporation experiments, 3 to 19% of the added radioactivity was detected in released {sup 14}CO{sub 2}, indicating that part of the added fatty acids were oxidatively decomposed. Partially purified fatty acid synthetase produced saturated and unsaturated fatty acids with chain lengths of C10 to C18. These results indicated that both aerobic and anaerobic mechanisms for the synthesis of unsaturated fatty acid are operating in this bacterium.

  13. Effect of propionic acid on fatty acid oxidation and ureagenesis.

    PubMed

    Glasgow, A M; Chase, H P

    1976-07-01

    Propionic acid significantly inhibited 14CO2 production from [1-14C] palmitate at a concentration of 10 muM in control fibroblasts and 100 muM in methylmalonic fibroblasts. This inhibition was similar to that produced by 4-pentenoic acid. Methylmalonic acid also inhibited 14CO2 production from [1-14C] palmitate, but only at a concentration of 1 mM in control cells and 5 mM in methylmalonic cells. Propionic acid (5 mM) also inhibited ureagenesis in rat liver slices when ammonia was the substrate but not with aspartate and citrulline as substrates. Propionic acid had no direct effect on either carbamyl phosphate synthetase or ornithine transcarbamylase. These findings may explain the fatty degeneration of the liver and the hyperammonemia in propionic and methylmalonic acidemia. PMID:934734

  14. 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.

  15. Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

    1974-01-01

    Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.

  16. Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

    PubMed Central

    Härtlein, M; Madern, D; Leberman, R

    1987-01-01

    Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli. Images PMID:3029694

  17. Mutational Separation of Aminoacylation and Cytokine Activities of Human Tyrosyl-tRNA Synthetase

    PubMed Central

    Kapoor, Mili; Otero, Francella J.; Slike, Bonnie M.; Ewalt, Karla L.; Yang, Xiang-Lei

    2009-01-01

    SUMMARY Aminoacyl-tRNA synthetases are known for catalysis of aminoacylation. Significantly, some mammalian synthetases developed cytokine functions possibly linked to disease-causing mutations in tRNA synthetases. Not understood is how epitopes for cytokine signaling were introduced into catalytic scaffolds without disturbing aminoacylation. Here we investigate human tyrosyl-tRNA synthetase, where a catalytic-domain surface helix—next to the active site—was recruited for IL-8-like cytokine signaling. Taking advantage of our high-resolution structure, the reciprocal impact of rational mutations designed to disrupt aminoacylation or cytokine signaling was investigated with multiple assays. The collective analysis demonstrated a protective fine–structure separation of aminoacylation from cytokine activities within the conserved catalytic domain. As a consequence, disease-causing mutations affecting cell signaling can arise without disturbing aminoacylation. These results with TyrRS also predict the previously unknown binding conformation of IL-8-like CXC cytokines. PMID:19477417

  18. Induction of angiogenesis by a fragment of human tyrosyl-tRNA synthetase.

    PubMed

    Wakasugi, Keisuke; Slike, Bonnie M; Hood, John; Ewalt, Karla L; Cheresh, David A; Schimmel, Paul

    2002-06-01

    The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis. PMID:11956181

  19. Mutational separation of aminoacylation and cytokine activities of human tyrosyl-tRNA synthetase.

    PubMed

    Kapoor, Mili; Otero, Francella J; Slike, Bonnie M; Ewalt, Karla L; Yang, Xiang-Lei

    2009-05-29

    Aminoacyl tRNA synthetases are known for catalysis of aminoacylation. Significantly, some mammalian synthetases developed cytokine functions possibly linked to disease-causing mutations in tRNA synthetases. Not understood is how epitopes for cytokine signaling were introduced into catalytic scaffolds without disturbing aminoacylation. Here we investigate human tyrosyl-tRNA synthetase, where a catalytic-domain surface helix, next to the active site, was recruited for interleukin-8-like cytokine signaling. Taking advantage of our high resolution structure, the reciprocal impact of rational mutations designed to disrupt aminoacylation or cytokine signaling was investigated with multiple assays. The collective analysis demonstrated a protective fine-structure separation of aminoacylation from cytokine activities within the conserved catalytic domain. As a consequence, disease-causing mutations affecting cell signaling can arise without disturbing aminoacylation. These results with TyrRS also predict the previously unknown binding conformation of interleukin-8-like CXC cytokines. PMID:19477417

  20. Nodule-specific modulation of glutamine synthetase in transgenic Medicago truncatula leads to inverse alterations in asparagine synthetase expression.

    PubMed

    Carvalho, Helena G; Lopes-Cardoso, Inês A; Lima, Ligia M; Melo, Paula M; Cullimore, Julie V

    2003-09-01

    Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed. PMID:12970490

  1. Nodule-Specific Modulation of Glutamine Synthetase in Transgenic Medicago truncatula Leads to Inverse Alterations in Asparagine Synthetase Expression1

    PubMed Central

    Carvalho, Helena G.; Lopes-Cardoso, Inês A.; Lima, Ligia M.; Melo, Paula M.; Cullimore, Julie V.

    2003-01-01

    Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed. PMID:12970490

  2. Lack of protective effect of thromboxane synthetase inhibitor (CGS-13080) on single dose radiated canine intestine

    SciTech Connect

    Barter, J.F.; Marlow, D.; Kamath, R.K.; Harbert, J.; Torrisi, J.R.; Barnes, W.A.; Potkul, R.K.; Newsome, J.T.; Delgado, G. )

    1991-03-01

    The effect of a thromboxane A2 synthetase inhibitor (CGS-13080) on canine intestine was studied using a single dose of radiation, and radioactive microspheres were used to determine resultant blood flow. Thromboxane A2 causes vasospasm and platelet aggregation and may play a dominant role in radiation injury. However, there was no effect on the intestinal blood flow diminution occurring after radiation in this laboratory model using this thromboxane A2 synthetase inhibitor.

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

    PubMed

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

    2014-04-01

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

  4. Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi

    SciTech Connect

    Byers, D.M.

    1989-01-01

    Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from (1-14C)myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from (14C)C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from (14C)acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development.

  5. The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence

    PubMed Central

    Avila-Ospina, Liliana; Marmagne, Anne; Talbotec, Joël; Krupinska, Karin; Masclaux-Daubresse, Céline

    2015-01-01

    Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions. PMID:25697791

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

    SciTech Connect

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

    2009-04-30

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

  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. Gain-of-function mutational activation of human tRNA synthetase procytokine.

    PubMed

    Yang, Xiang-Lei; Kapoor, Mili; Otero, Francella J; Slike, Bonnie M; Tsuruta, Hiro; Frausto, Ricardo; Bates, Alison; Ewalt, Karla L; Cheresh, David A; Schimmel, Paul

    2007-12-01

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases. PMID:18096501

  9. Trans Fatty Acids

    NASA Astrophysics Data System (ADS)

    Doyle, Ellin

    1997-09-01

    Fats and their various fatty acid components seem to be a perennial concern of nutritionists and persons concerned with healthful diets. Advice on the consumption of saturated, polyunsaturated, monounsaturated, and total fat bombards us from magazines and newspapers. One of the newer players in this field is the group of trans fatty acids found predominantly in partially hydrogenated fats such as margarines and cooking fats. The controversy concerning dietary trans fatty acids was recently addressed in an American Heart Association (AHA) science advisory (1) and in a position paper from the American Society of Clinical Nutrition/American Institute of Nutrition (ASCN/AIN) (2). Both reports emphasize that the best preventive strategy for reducing risk for cardiovascular disease and some types of cancer is a reduction in total and saturated fats in the diet, but a reduction in the intake of trans fatty acids was also recommended. Although the actual health effects of trans fatty acids remain uncertain, experimental evidence indicates that consumption of trans fatty acids adversely affects serum lipid levels. Since elevated levels of serum cholesterol and triacylglycerols are associated with increased risk of cardiovascular disease, it follows that intake of trans fatty acids should be minimized.

  10. Mammalian Fatty Acid Elongases

    PubMed Central

    Jump, Donald B.

    2009-01-01

    Summary Very long chain fatty acids confer functional diversity on cells by variations in their chain length and degree of unsaturation. Microsomal fatty acid elongation represents the major pathway for determining the chain length of saturated, monounsaturated, and polyunsaturated fatty acids in cellular lipids. The overall reaction for fatty acid elongation involves four enzymes and utilizes malonyl CoA, NADPH, and fatty acyl CoA as substrates. While the fundamental pathway and its requirements have been known for many years, recent advances have revealed a family of enzymes involved in the first step of the reaction, i.e., the condensation reaction. Seven fatty acid elongase subtypes (Elovl #1–7) have been identified in the mouse, rat, and human genomes. These enzymes determine the rate of overall fatty acid elongation. Moreover, these enzymes also display differential substrate specificity, tissue distribution, and regulation, making them important regulators of cellular lipid composition as well as specific cellular functions. Herein, methods are described to measure elongase activity, analyze elongation products, and alter cellular elongase expression. PMID:19763486

  11. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  12. Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids.

    PubMed Central

    Taylor, F; Cronan, J E

    1976-01-01

    Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-[methyl-3H]methionine suicide procedure. Two mutants were isolated. Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain. The CFA deficiency is attributed to a deficiency of CFA synthetase activity. Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain. Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts. We have not yet detected a physiological phenotype for these mutants. These strains grow normally at various temperatures in a variety of media. We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions. The survival of both mutants is identical to that of the parental strain under all conditions tested. The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain. The transport of several amino acids also seems normal in these mutants. PMID:1107324

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6 (encoding a very-long-chain ACS), a human homolog of bgm and dbb. PMID:26893370

  14. Levels of acyl-coenzyme A synthetase 5 in urothelial cells and corresponding neoplasias reflect cellular differentiation.

    PubMed

    Gaisa, Nadine T; Reinartz, Andrea; Schneider, Ursula; Klaus, Christina; Heidenreich, Axel; Jakse, Gerhard; Kaemmerer, Elke; Klinkhammer, Barbara Mara; Knuechel, Ruth; Gassler, Nikolaus

    2013-03-01

    Metabolic components like fatty acids and acyl-Coenzyme A (acyl-CoA) thioesters have been implicated in the pathogenesis of various tumours. The activation of fatty acids to acyl-CoAs is catalysed by long chain acyl-CoA synthetases (ACSLs), and impairment of ACSL expression levels has been associated with tumourigenesis and progression. Since ACSLs have never been investigated in bladder tissues, the study aims to characterize ACSL expression and acyl-CoA synthesis in normal and neoplastic bladder tissues, as well as cell lines. ACSL isoforms 1, 3, 4 and 5 and synthesis of acyl-CoAs were analysed using qRT-PCR, western blot analysis, immunohistochemistry and lipid mass spectrometry. In normal urothelium, expression of ACSL1, 3, 4 and 5, with highest levels of ACSL isoform 5 was found. However, ACSL5 expression was reduced in corresponding neoplastic tissues and urothelial cell lines depending on the grade of cellular differentiation. Anti-ACSL5 immunostainings showed expression in normal urothelium and a gradual loss of ACSL5 protein via pre-invasive lesions to invasive carcinomas. High expression of ACSL5 correlated with increased α-galactosidase activity and positive Uroplakin III staining in tumours. In contrast, synthesis of acyl-CoAs was enhanced in neoplastic bladder tissues compared to normal urothelium, and reflected an increase with respect to cellular differentiation. These results confirm an expression of ACSLs, especially isoform 5, in human urothelium, prove enzymatic/lipidomic changes in bladder cancer tissues, and suggest an involvement of ACSL5 in cellular maturation and/or senescence with possible effects onto induction of tumour formation or progression. Further work may identify responsible pathway alterations, and attempting to re-balance the metabolic equilibrium of the urothelium may offer a further opportunity for tumour treatment and prevention. PMID:23348389

  15. 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

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

    PubMed

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

    2016-04-01

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

  17. Long-Chain Acyl CoA Synthetase 4A regulates Smad activity and dorsoventral patterning in the zebrafish embryo

    PubMed Central

    Miyares, Rosa Linda; Stein, Cornelia; Renisch, Björn; Anderson, Jennifer Lynn; Hammerschmidt, Matthias; Farber, Steven Arthur

    2013-01-01

    Summary Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated Bmp signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 MAPK and the Akt-mediated inhibition of glycogen synthase kinase 3 (GSK3), critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes. PMID:24332754

  18. Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide.

    PubMed

    Mashima, Tetsuo; Sato, Shigeo; Okabe, Sachiko; Miyata, Satoshi; Matsuura, Masaaki; Sugimoto, Yoshikazu; Tsuruo, Takashi; Seimiya, Hiroyuki

    2009-08-01

    Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide. PMID:19459852

  19. Peroxisome proliferator-activated receptor gamma 2 and acyl-CoA synthetase 5 polymorphisms influence diet response.

    PubMed

    Adamo, Kristi B; Dent, Robert; Langefeld, Carl D; Cox, Miranda; Williams, Kathryn; Carrick, Kevin M; Stuart, Joan S; Sundseth, Scott S; Harper, Mary-Ellen; McPherson, Ruth; Tesson, Frédérique

    2007-05-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness. PMID:17495181

  20. 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

  1. Fatty liver - nonalcoholic

    MedlinePlus

    ... American Gastroenterological Association. The diagnosis and management of non-alcoholic fatty liver disease: Practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and ...

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

    SciTech Connect

    Webb, G.C.; Vaska, V.L.; Ford, J.H.

    1995-12-10

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

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

    PubMed Central

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

    2014-01-01

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

  4. Role of bioactive fatty acids in nonalcoholic fatty liver disease.

    PubMed

    Juárez-Hernández, Eva; Chávez-Tapia, Norberto C; Uribe, Misael; Barbero-Becerra, Varenka J

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by fat deposition in hepatocytes, and a strong association with nutritional factors. Dietary fatty acids are classified according to their biochemical properties, which confer their bioactive roles. Monounsaturated fatty acids have a dual role in various human and murine models. In contrast, polyunsaturated fatty acids exhibit antiobesity, anti steatosic and anti-inflammatory effects. The combination of these forms of fatty acids-according to dietary type, daily intake and the proportion of n-6 to n-3 fats-can compromise hepatic lipid metabolism. A chemosensory rather than a nutritional role makes bioactive fatty acids possible biomarkers for NAFLD. Bioactive fatty acids provide health benefits through modification of fatty acid composition and modulating the activity of liver cells during liver fibrosis. More and better evidence is necessary to elucidate the role of bioactive fatty acids in nutritional and clinical treatment strategies for patients with NAFLD. PMID:27485440

  5. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue. PMID:25801336

  6. Glutamine synthetase predicts adjuvant TACE response in hepatocellular carcinoma

    PubMed Central

    Zhang, Bo; Liu, Kai; Zhang, Jian; Dong, Liwei; Jin, Zhichao; Zhang, Xinji; Xue, Feng; He, Jia

    2015-01-01

    Background: Adjuvant transcatheter arterial chemoembolization (TACE) is associated with better outcome and reduced tumor recurrence in hepatocellular carcinoma (HCC) patients. This study aimed to investigate the relationship between glutamine synthetase (GS) expression and survival of HCC patients after postoperative adjuvant TACE. Methods: We retrospectively analyzed 554 HCC patients in two independent cohorts who underwent curative resection. Immunohistochemistry assay was used to investigate the expression of GS protein and evaluate the association with survival and the response to adjuvant TACE. Results: In training cohort, patients with low GS expression who received postoperative adjuvant TACE showed a better overall survival (OS) (P<0.001) and less early phase recurrence (P=0.016). Adjuvant TACE was an independent prognostic factor for 5-year OS (HR=0.408, 95% CI 0.261-0.639, P<0.001) and early phase recurrence (HR=0.592, 95% CI 0.376-0.931, P=0.023). The same result was confirmed in validation cohort. Patients with high GS expression in both cohorts did not have a significant response to adjuvant TACE in OS and early phase recurrence. Conclusions: GS status in tumor might be a useful tool in the selection of HCC patients who would be likely to benefit from postoperative adjuvant TACE. PMID:26884995

  7. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold

    PubMed Central

    Dong, Shi-Hui; Tang, Weixin; Lukk, Tiit; Yu, Yi; Nair, Satish K; van der Donk, Wilfred A

    2015-01-01

    The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin. DOI: http://dx.doi.org/10.7554/eLife.07607.001 PMID:26226635

  8. The aminoacyl-tRNA synthetases of Drosophila melanogaster

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Chen, Q; Silflow, C D

    1996-11-01

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

  10. 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

  11. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    Das, S.; Gillin, F.D.

    1987-05-01

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

  12. Evidence for allosteric regulation of succinyl-CoA synthetase.

    PubMed Central

    Um, H D; Klein, C

    1993-01-01

    We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5'-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8240297

  13. Evidence for allosteric regulation of succinyl-CoA synthetase.

    PubMed

    Um, H D; Klein, C

    1993-11-01

    We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5'-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites. PMID:8240297

  14. Cloning of the glutamine synthetase gene from group B streptococci.

    PubMed

    Suvorov, A N; Flores, A E; Ferrieri, P

    1997-01-01

    The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed. PMID:8975911

  15. Adenine nucleotides as allosteric effectors of PEA seed glutamine synthetase

    SciTech Connect

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

    1986-05-01

    The energy charge in the plant cell has been proposed as a regulator of glutamine synthetase (GS) activity. The authors have shown that 2.1 moles of ..gamma..(/sup 32/P)-ATP were bound/mole subunits of purified pea seed GS during complete inactivation with methionine sulfoximine. Since GS has one active site per subunit, the second binding site provides the potential for allosteric regulation of GS by adenine nucleotides. The authors have investigated the inhibition of the ATP-dependent synthetic activity by ADP and AMP. ADP and AMP cannot completely inhibit GS; but ATP does overcome the inhibition by ADP and AMP as shown by plots of % inhibition vs inhibitor concentration. This indicates that inhibition of GS by ADP or AMP is not completely due to competitive inhibition. In the absence of ADP or AMP, double reciprocal plots for ATP are linear below 10 mM; however, in the presence of either ADP or AMP these pots are curvilinear downwards. The ratio of Vm/asymptote is less than 1. The Hill number for ATP in the absence of ADP or AMP is 0.93 but decreases with increasing ADP or AMP to a value of 0.28 with 10 mM ADP. These data are consistent with negative cooperativity by ADP and AMP. Thus, as the ADP/ATP or AMP/ATP ratios are increased GS activity decreases. This is consistent with regulation of GS activity by energy charge in planta.

  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. PMID:25896882

  17. Is acetylcarnitine a substrate for fatty acid synthesis in plants

    SciTech Connect

    Roughan, G. ); Post-Beittenmiller, D.; Ohlrogge, J. ); Browse, J. )

    1993-04-01

    Long-chain fatty acid synthesis from [1-[sup 14]C]acetylcarnitine by chloroplasts isolated from spinach (Spinacia oleracea), pea (Pisum sativum), amaranthus (Amaranthus lividus), or maize (Zea mays) occurred at less than 2% of the rate of fatty acid synthesis from [1-[sup 14]C]acetate irrespective of the maturity of the leaves or whether the plastids were purified using sucrose or Percoll medium. [1-[sup 14]C]Acetylcarnitine was not significantly utilized by highly active chloroplasts rapidly prepared from pea and spinach using methods not involving density gradient centrifugation. [1-[sup 14]C]Acetylcarnitine was recovered quantitatively from chloroplast incubations following 10 min in the light. Unlabeled acetyl-L-carnitine (0.4 mM) did not compete with [1-[sup 14]C]acetate (0.2 mM) as a substrate for fatty acid synthesis by any of the more than 70 chloroplast preparations tested in this study. Carnitine acetyltransferase activity was not detected in any chloroplast preparation and was present in whole leaf homogenates at about 0.1% of the level of acetyl-coenzyme A synthetase activity. When supplied to detached pea shoots and detached spinach, amaranthus, and maize leaves via the transpiration stream, 1 to 4% of the [1-[sup 14]C]acetylcarnitine and 47 to 57% of the [1-[sup 14]C]acetate taken up was incorporated into lipids. Most (78--82%) of the [1-[sup 14]C]acetylcarnitine taken up was recovered intact. It is concluded that acetylcarnitine is not a major precursor for fatty acid synthesis in plants. 29 refs., 5 tabs.

  18. Cyclic AMP-dependent Protein Lysine Acylation in Mycobacteria Regulates Fatty Acid and Propionate Metabolism*

    PubMed Central

    Nambi, Subhalaxmi; Gupta, Kallol; Bhattacharyya, Moitrayee; Ramakrishnan, Parvathy; Ravikumar, Vaishnavi; Siddiqui, Nida; Thomas, Ann Terene; Visweswariah, Sandhya S.

    2013-01-01

    Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host. PMID:23553634

  19. Proteomic identification of glutamine synthetase as a differential marker for oligodendrogliomas and astrocytomas

    PubMed Central

    Zhuang, Zhengping; Qi, Meng; Li, Jie; Okamoto, Hiroaki; Xu, David S.; Iyer, Rajiv R.; Lu, Jie; Yang, Chunzhang; Weil, Robert J.; Vortmeyer, Alexander; Lonser, Russell R.

    2016-01-01

    Object Astrocytomas and oligodendrogliomas are primary CNS tumors that remain a challenge to differentiate histologically because of their morphological variability and because there is a lack of reliable differential diagnostic markers. To identify proteins that are differentially expressed between astrocytomas and oligodendrogliomas, the authors analyzed the proteomic expression patterns and identified uniquely expressed proteins in these neoplasms. Methods Proteomes of astrocytomas and oligodendrogliomas were analyzed using 2D gel electrophoresis and subsequent computerized gel analysis to detect differentially expressed proteins. The proteins were identified using high-performance liquid chromatography accompanied by tandem mass spectrometry. To determine the role of the differentially expressed proteins in astrocytes, undifferentiated glial cell cultures were treated with dibutyryl–cyclic adenosine monophosphate (cAMP). Results Two-dimensional gel electrophoresis revealed that glutamine synthetase was differentially expressed in astrocytomas and oligodendrogliomas. Western blot and immunohistochemical analyses confirmed the increased expression of glutamine synthetase in astrocytomas compared with oligodendrogliomas. Whereas glutamine synthetase expression was demonstrated across all grades of astrocytomas (Grade II–IV [15 tumors]) and oligoastrocytomas (4 tumors), it was expressed in only 1 oligodendroglioma (6% [16 tumors]). Treatment of undifferentiated glial cell cultures with dibutyryl-cAMP resulted in astrocyte differentiation that was associated with increased levels of glial fibrillary acidic protein and glutamine synthetase. Conclusions These data indicate that glutamine synthetase expression can be used to distinguish astrocytic from oligodendroglial tumors and may play a role in the pathogenesis of astrocytomas. PMID:21682567

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

    PubMed

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

    2016-08-19

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

  1. Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

    SciTech Connect

    Taylor, G.R.; Barclay, B.J.; Storms, R.K.; Friesen, J.D.; Haynes, R.H.

    1982-04-01

    The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp/sup +/ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy/sup +/ transformants directly, it was found that all pTL1 transformants were phenotypically Thy/sup +/ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of (6-/sup 3/H)dUMP to (6-/sup 3/H)dTMP. In protein extracts from the thymidylate auxotroph (tmpl-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

  2. A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

    PubMed Central

    Cerini, C; Kerjan, P; Astier, M; Gratecos, D; Mirande, M; Sémériva, M

    1991-01-01

    In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed. Images PMID:1756734

  3. Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A

    PubMed Central

    Manhas, Reetika; Tripathi, Pankaj; Khan, Sameena; Sethu Lakshmi, Bhavana; Lal, Shambhu Krishan; Gowri, Venkatraman Subramanian; Sharma, Amit; Madhubala, Rentala

    2014-01-01

    Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target. PMID:24610810

  4. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

    PubMed Central

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P.; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed. PMID:26270653

  5. Membrane Anchoring of Aminoacyl-tRNA Synthetases by Convergent Acquisition of a Novel Protein Domain*

    PubMed Central

    Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Ribas de Pouplana, Lluis; Luque, Ignacio

    2011-01-01

    Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain. PMID:21965654

  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. Structural plasticity of an aminoacyl-tRNA synthetase active site

    PubMed Central

    Turner, James M.; Graziano, James; Spraggon, Glen; Schultz, Peter G.

    2006-01-01

    Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substrate-bound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the α8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyl-tRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids. PMID:16618920

  8. 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

  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. PMID:27270277

  10. Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.

    PubMed Central

    Moran, R G; Mulkins, M; Heidelberger, C

    1979-01-01

    Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell. PMID:160558

  11. Pathogenic implications of human mitochondrial aminoacyl-tRNA synthetases.

    PubMed

    Schwenzer, Hagen; Zoll, Joffrey; Florentz, Catherine; Sissler, Marie

    2014-01-01

    Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders. PMID:23824528

  12. Proteasomal degradation of glutamine synthetase regulates schwann cell differentiation.

    PubMed

    Saitoh, Fuminori; Araki, Toshiyuki

    2010-01-27

    Rapid saltatory nerve conduction is facilitated by myelin structure, which is composed of Schwann cells in the peripheral nervous system. Schwann cells drastically change their phenotype following peripheral nerve injury. These phenotypic changes are required for efficient degeneration/regeneration. We previously identified ZNRF1 as an E3 ubiquitin ligase containing a RING finger motif, whose expression is upregulated in the Schwann cells following nerve injury. This suggested that posttranscriptional regulation of protein expression in Schwann cells may be involved in their phenotypic changes during nerve degeneration/regeneration. Here we report the identification of glutamine synthetase (GS), an enzyme that synthesizes glutamine using glutamate and ammonia, as a substrate for E3 activity of ZNRF1 in Schwann cells. GS is known to be highly expressed in differentiated Schwann cells, but its functional significance has remained unclear. We found that during nerve degeneration/regeneration, GS expression is controlled mostly by ZNRF1-dependent proteasomal degradation. We also found that Schwann cells increase oxidative stress upon initiation of nerve degeneration, which promotes carbonylation and subsequent degradation of GS. Surprisingly, we discovered that GS expression regulates Schwann cell differentiation; i.e., increased GS expression promotes myelination via its enzymatic activity. Among the substrates and products of GS, increased glutamate concentration inhibited myelination and yet promoted Schwann cell proliferation by activating metabotropic glutamate receptor signaling. This would suggest that GS may exert its effect on Schwann cell differentiation by regulating glutamate concentration. These results indicate that the ZNRF1-GS system may play an important role in correlating Schwann cell metabolism with its differentiation. PMID:20107048

  13. 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.

  14. 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.

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

    DOEpatents

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

    2009-05-05

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

  16. 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.

  17. 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. PMID:26984560

  18. Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

    PubMed Central

    Billich, Andreas; Zocher, Rainer

    1988-01-01

    The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase. Images PMID:16347758

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

    PubMed Central

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

    2010-01-01

    Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs, essential substrates for accurate protein synthesis. Beyond their central role in translation some of these enzymes or their orthologs are recruited for alternative functions, not always related to their primary cellular role. We investigate here the enzymatic properties of GenX (also called PoxA and YjeA), an ortholog of bacterial class II lysyl-tRNA synthetase. GenX is present in most Gram-negative bacteria and is homologous to the catalytic core of lysyl-tRNA synthetase, but it lacks the amino terminal anticodon binding domain of the latter enzyme. We show that, in agreement with its well-conserved lysine binding site, GenX can activate in vitro L-lysine and lysine analogs, but does not acylate tRNALys or other cellular RNAs. PMID:20580719

  20. Variations in the Localization of Acetyl-Coenzyme A Synthetase in Aerobic Yeast Cells

    PubMed Central

    Klein, Harold P.; Jahnke, Linda

    1971-01-01

    In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected. Images PMID:4102333

  1. Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme.

    PubMed Central

    Dean, D R; Hoch, J A; Aronson, A I

    1977-01-01

    A mutational leading to glutamine auxotrophy was located near a 5-fluorouracil resistance marker in the citB-thyA region of the Bacillus subtilis chromosome. This mutation resulted in a glutamine synthetase with altered kinetic and feedback properties. The specific activity of manganese-stimulated glutamine synthetase activity in crude extracts was 18-fold higher, and the magnesium-stimulated activity was about 30% that of the wild type. Quantitation of the enzyme by precipitation with antibody prepared against pure enzyme confirmed the presence of high enzyme levels in the mutant. This mutation is very closely linked (recombination index of 0.03) to another glutamine auxotroph containing enzyme with altered electrophoretic and heat sensitivity properties. Mutations in the structural gene for glutamine synthetase may result not only in altered catalytic and regulatory properties but also in altered production of the enzyme. Images PMID:19424

  2. Isolation and characterisation of a ferrirhodin synthetase gene from the sugarcane pathogen Fusarium sacchari.

    PubMed

    Munawar, Asifa; Marshall, James W; Cox, Russell J; Bailey, Andy M; Lazarus, Colin M

    2013-02-11

    FSN1, a gene isolated from the sugar-cane pathogen Fusarium sacchari, encodes a 4707-residue nonribosomal peptide synthetase consisting of three complete adenylation, thiolation and condensation modules followed by two additional thiolation and condensation domain repeats. This structure is similar to that of ferricrocin synthetase, which makes a siderophore that is involved in intracellular iron storage in other filamentous fungi. Heterologous expression of FSN1 in Aspergillus oryzae resulted in the accumulation of a secreted metabolite that was identified as ferrirhodin. This siderophore was found to be present in both mycelium and culture filtrates of F. sacchari, whereas ferricrocin is found only in the mycelium, thus suggesting that ferricrocin is an intracellular storage siderophore in F. sacchari, whereas ferrirhodin is used for iron acquisition. To our knowledge, this is the first report to characterise a ferrirhodin synthetase gene functionally. PMID:23307607

  3. A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.

    PubMed

    Wakasugi, Keisuke; Slike, Bonnie M; Hood, John; Otani, Atsushi; Ewalt, Karla L; Friedlander, Martin; Cheresh, David A; Schimmel, Paul

    2002-01-01

    Aminoacyl-tRNA synthetases catalyze the first step of protein synthesis. It was shown recently that human tyrosyl-tRNA synthetase (TyrRS) can be split into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. Tryptophanyl-tRNA synthetase (TrpRS) is a close homologue of TyrRS. A natural fragment, herein designated as mini TrpRS, was shown by others to be produced by alternative splicing. Production of this fragment is reported to be stimulated by IFN-gamma, a cytokine that also stimulates production of angiostatic factors. Mini TrpRS is shown here to be angiostatic in a mammalian cell culture system, the chicken embryo, and two independent angiogenesis assays in the mouse. The full-length enzyme is inactive in the same assays. Thus, protein synthesis may be linked to the regulation of angiogenesis by a natural fragment of TrpRS. PMID:11773626

  4. A human aminoacyl-tRNA synthetase as a regulator of angiogenesis

    PubMed Central

    Wakasugi, Keisuke; Slike, Bonnie M.; Hood, John; Otani, Atsushi; Ewalt, Karla L.; Friedlander, Martin; Cheresh, David A.; Schimmel, Paul

    2002-01-01

    Aminoacyl-tRNA synthetases catalyze the first step of protein synthesis. It was shown recently that human tyrosyl-tRNA synthetase (TyrRS) can be split into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. Tryptophanyl-tRNA synthetase (TrpRS) is a close homologue of TyrRS. A natural fragment, herein designated as mini TrpRS, was shown by others to be produced by alternative splicing. Production of this fragment is reported to be stimulated by IFN-γ, a cytokine that also stimulates production of angiostatic factors. Mini TrpRS is shown here to be angiostatic in a mammalian cell culture system, the chicken embryo, and two independent angiogenesis assays in the mouse. The full-length enzyme is inactive in the same assays. Thus, protein synthesis may be linked to the regulation of angiogenesis by a natural fragment of TrpRS. PMID:11773626

  5. Omega-3 fatty acids (image)

    MedlinePlus

    Omega-3 fatty acids are a form of polyunsaturated fat that the body derives from food. Omega-3s (and omega-6s) are known as essential fatty acids (EFAs) because they are important for good health. ...

  6. Discovery of essential fatty acids

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2015-01-01

    Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research. PMID:25339684

  7. Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

    PubMed

    Palacios, R; Campomanes, M; Quinto, C

    1977-05-10

    The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose. PMID:16013

  8. Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

    PubMed Central

    Anderson, J. W.; Fowden, L.

    1970-01-01

    1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

  9. Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae.

    PubMed

    Leber, Christopher; Polson, Brian; Fernandez-Moya, Ruben; Da Silva, Nancy A

    2015-03-01

    The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes. β-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490mg/L. Combining these two disrupted pathways, a sextuple mutant (Δfaa1 Δfaa4 Δfat1 Δfaa2 Δpxa1 Δpox1) was able to produce 1.3g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1, TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae. PMID:25461829

  10. Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A).

    PubMed

    Fontes, R; Günther Sillero, M A; Sillero, A

    1999-03-01

    The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant. PMID:10385004

  11. Acute Onset Anti-Synthetase Syndrome With Pericardial Effusion and Non-Specific Interstitial Pneumonia

    PubMed Central

    Shah, Aditya; Patel, Samir R.

    2016-01-01

    Anti-synthetase syndrome (AS) is a clinical entity which is described classically by the triad of interstitial lung disease (ILD), inflammatory myositis and presence of aminoacyl-tRNA synthetase antibodies (ASA). We describe a rare presentation of this condition with regard to the uncharacteristically acute nature of presentation, acute decompensation in clinical condition, development of acute interstitial pneumonitis requiring rescue extracorporeal membrane oxygenation (ECMO) and accompaniment of significant pericardial effusion on presentation, followed by rapid improvement with initiation of steroids. PMID:27540445

  12. Mutants of Phycomyces blakesleeanus Defective in Acetyl-CoA Synthetase

    PubMed

    Garre; Torres-Martinez

    1996-03-01

    Nine mutants of the filamentous fungus Phycomyces blakesleeanus have been isolated on the basis of their resistance to fluoroacetate. None of the isolates uses acetate as the sole carbon source. Genetic complementation experiments revealed that all the mutants belong to the same complementation group. Biochemical analysis indicated that the acetate-induced acetyl-CoA synthetase activity is abolished in all nine mutants, thus suggesting that they are affected in the gene coding for acetyl-CoA synthetase (facA). PMID:8812287

  13. Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus.

    PubMed

    Garre, V; Murillo, F J; Torres-Martínez, S

    1994-08-01

    A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanus protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA- mutation of this organism. In P. blakesleeanus, the expression of facA is induced by acetate. PMID:7914670

  14. Acute Onset Anti-Synthetase Syndrome With Pericardial Effusion and Non-Specific Interstitial Pneumonia.

    PubMed

    Shah, Aditya; Patel, Samir R

    2016-09-01

    Anti-synthetase syndrome (AS) is a clinical entity which is described classically by the triad of interstitial lung disease (ILD), inflammatory myositis and presence of aminoacyl-tRNA synthetase antibodies (ASA). We describe a rare presentation of this condition with regard to the uncharacteristically acute nature of presentation, acute decompensation in clinical condition, development of acute interstitial pneumonitis requiring rescue extracorporeal membrane oxygenation (ECMO) and accompaniment of significant pericardial effusion on presentation, followed by rapid improvement with initiation of steroids. PMID:27540445

  15. Fatty Acids of Thiobacillus thiooxidans

    PubMed Central

    Levin, Richard A.

    1971-01-01

    Fatty acid spectra were made on Thiobacillus thiooxidans cultures both in the presence and absence of organic compounds. Small additions of glucose or acetate had no significant effect either on growth or fatty acid content. The addition of biotin had no stimulatory effect but did result in slight quantitative changes in the fatty acid spectrum. The predominant fatty acid was a C19 cyclopropane acid. PMID:4945206

  16. Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

    PubMed Central

    Davies, R C; Neuberger, A

    1979-01-01

    1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active. PMID:312102

  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. New bioactive fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many oxygenated fatty acids are bioactive compounds. Nocardia cholesterolicum and Flavobacterium DS5 convert oleic acid to 10 hydroxy stearic acid and linoleic acid to 10-hydroxy-12(Z)-octadecanoic acid. Pseudomonas aeruginosa PR3 converts oleic acid to the new compounds, 7,10-dihydroxy-8(E)-octad...

  19. New Bioactive Fatty Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many oxygenated fatty acids are bioactive compounds. Nocardia cholesterolicum and Flavobacterium DS5 convert oleic acid to 10 hydroxy stearic acid and linoleic acid to 10-hydroxy-12(Z)-octadecanoic acid. Pseudomonas aeruginosa PR3 converts oleic acid to new compounds, 7,10-dihydroxy-8(E)-octadecen...

  20. Divergent anticodon recognition in contrasting glutamyl-tRNA synthetases.

    PubMed

    Lee, Joohee; Hendrickson, Tamara L

    2004-12-10

    The pathogenic bacterium Helicobacter pylori utilizes two essential glutamyl-tRNA synthetases (GluRS1 and GluRS2). These two enzymes are closely related in evolution and yet they aminoacylate contrasting tRNAs. GluRS1 is a canonical discriminating GluRS (D-GluRS) that biosynthesizes Glu-tRNA(Glu) and cannot make Glu-tRNA(Gln). In contrast, GluRS2 is non-canonical as it is only essential for the production of misacylated Glu-tRNA(Gln). The co-existence and evident divergence of these two enzymes was capitalized upon to directly examine how GluRS2 acquired tRNA(Gln) specificity. One key feature that distinguishes tRNA(Glu) from tRNA(Gln) is the third position in the anticodon of each tRNA (C36 versus G36, respectively). By comparing sequence alignments of different GluRSs, including GluRS1s and GluRS2s, to the crystal structure of the Thermus thermophilus D-GluRS:tRNA(Glu) complex, a divergent pattern of conservation in enzymes that aminoacylate tRNA(Glu)versus those specific for tRNA(Gln) emerged and was experimentally validated. In particular, when an arginine conserved in discriminating GluRSs and GluRS1s was inserted into Hp GluRS2 (Glu334Arg GluRS2), the catalytic efficiency of the mutant enzyme (k(cat)/K(Mapp)) was reduced by approximately one order of magnitude towards tRNA(Gln). However, this mutation did not introduce activity towards tRNA(Glu). In contrast, disruption of a glycine that is conserved in all GluRS2s but not in other GluRSs (Gly417Thr GluRS2) generated a mutant GluRS2 with weak activity towards tRNA(Glu1). Synergy between these two mutations was observed in the double mutant (Glu334Arg/Gly417Thr GluRS2), which specifically and more robustly aminoacylates tRNA(Glu1) instead of tRNA(Gln). As GluRS1 and GluRS2 are related by an apparent gene duplication event, these results demonstrate that we can experimentally map critical evolutionary events in the emergence of new tRNA specificities. PMID:15561136

  1. The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

    PubMed Central

    Saggerson, E. D.; Greenbaum, A. L.

    1970-01-01

    1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO2 and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [14C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of

  2. Microbial production of fatty alcohols.

    PubMed

    Fillet, Sandy; Adrio, José L

    2016-09-01

    Fatty alcohols have numerous commercial applications, including their use as lubricants, surfactants, solvents, emulsifiers, plasticizers, emollients, thickeners, and even fuels. Fatty alcohols are currently produced by catalytic hydrogenation of fatty acids from plant oils or animal fats. Microbial production of fatty alcohols may be a more direct and environmentally-friendly strategy since production is carried out by heterologous enzymes, called fatty acyl-CoA reductases, able to reduce different acyl-CoA molecules to their corresponding primary alcohols. Successful examples of metabolic engineering have been reported in Saccharomyces cerevisiae and Escherichia coli in which the production of fatty alcohols ranged from 1.2 to 1.9 g/L, respectively. Due to their metabolic advantages, oleaginous yeasts are considered the best hosts for production of fatty acid-derived chemicals. Some of these species can naturally produce, under specific growth conditions, lipids at high titers (>50 g/L) and therefore provide large amounts of fatty acyl-CoAs or fatty acids as precursors. Very recently, taking advantage of such features, over 8 g/L of C16-C18 fatty alcohols have been produced in Rhodosporidium toruloides. In this review we summarize the different metabolic engineering strategies, hosts and cultivation conditions used to date. We also point out some future trends and challenges for the microbial production of fatty alcohols. PMID:27465852

  3. Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.

    PubMed Central

    Rizzi, M; Nessi, C; Mattevi, A; Coda, A; Bolognesi, M; Galizzi, A

    1996-01-01

    NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction. The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases. Images PMID:8895556

  4. Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

    PubMed

    Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

    2015-12-01

    Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. PMID:26472928

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

    PubMed

    Cane, D E; Walsh, C T

    1999-12-01

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) catalyze chain elongation from simple building blocks to create a diverse array of natural products. PKS and NRPS proteins share striking architectural and organizational similarities that can be exploited to generate entirely new natural products. PMID:10631508

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

    PubMed Central

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

    2007-01-01

    Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

  7. Structure of Pyrrolysyl-tRNA Synthetase, an Archaeal Enzyme for Genetic Code Innovation

    SciTech Connect

    Kavran,J.; Gundllapalli, S.; O'Donoghue, P.; Englert, M.; Soll, D.; Steitz, T.

    2007-01-01

    Pyrrolysine (Pyl), the 22nd natural amino acid and genetically encoded by UAG, becomes attached to its cognate tRNA by pyrrolysyl-tRNA synthetase (PylRS). We have determined three crystal structures of the Methanosarcina mazei PylRS complexed with either AMP-PNP, Pyl-AMP plus pyrophosphate, or the Pyl analogue N-e-[(cylopentyloxy)carbonyl]-l-lysine plus ATP. The structures reveal that PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain. A comparison of these structures with previously determined class II tRNA synthetase complexes illustrates that different substrate specificities derive from changes in a small number of residues that form the substrate side-chain-binding pocket. The knowledge of these structures allowed the placement of PylRS in the aminoacyl-tRNA synthetase (aaRS) tree as the last known synthetase that evolved for genetic code expansion, as well as the finding that Pyl arose before the last universal common ancestral state. The PylRS structure provides an excellent framework for designing new aaRSs with altered amino acid specificity.

  8. 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

  9. Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

    PubMed Central

    Adul Rahman, R N; Jongsareejit, B; Fujiwara, S; Imanaka, T

    1997-01-01

    The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction. PMID:9172372

  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. Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1deficiency with N-carbamylglutamate

    PubMed Central

    Ah Mew, Nicholas; McCarter, Robert; Daikhin, Yevgeny; Lichter, Uta; Nissim, Ilana; Yudkoff, Marc; Tuchman, and Mendel

    2014-01-01

    Identical studies employing stable isotopes were performed before and after a 3-day trial of oral N-carbamylglutamate (NCG) in 5 subjects with late onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in one subject. PMID:24880889

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

    PubMed

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

    2016-01-14

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

  13. Gain-of-Function Mutational Activation of Human tRNA Synthetase Procytokine

    PubMed Central

    Yang, Xiang-Lei; Kapoor, Mili; Otero, Francella J.; Slike, Bonnie M.; Tsuruta, Hiro; Frausto, Ricardo; Bates, Alison; Ewalt, Karla L.; Cheresh, David A.; Schimmel, Paul

    2008-01-01

    Summary Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain-of-function. Native tRNA synthetases, like TyrRS and TrpRS, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis we hypothesized that a steric block of a critical ELR motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases. PMID:18096501

  14. DISTRIBUTION OF THE GLUTAMINE SYNTHETASE ISOZYME GSP1 IN MAIZE (ZEA MAYS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Higher plants contain families of glutamine synthetase (GS) isozymes. In maize (Zea mays L.), GSp1, the predominant GS isozyme of the developing kernel, is abundant in the pedicel and pericarp, but absent from the endosperm and embryo. Determination of GSp1 tissue distribution in vegetative tissue...

  15. Synthesis and activities of branched-chain aminoacyl-tRNA synthetases in threonine deaminase mutants of Escherichia coli.

    PubMed Central

    Williams, A L; Whitfield, S M; Williams, L S

    1978-01-01

    Valyl-, isoleucyl-, and leucyl-tRNA synthetase activities were examined in an Escherichia coli K-12 strain that possessed a deletion of three genes of the ilv gene cluster, ilvD, A, and C, and in a strain with the same deletion that also carried the lambdadilvCB bacteriophage. It was observed that the branched-chain tRNA synthetase activities of both strains were considerably less than those of the normal strain during growth in unrestricted medium. Furthermore, during an isoleucine limitation, there was a further reduction in isoleucyl-tRNA synthetase activity and an absence of the isoleucine-mediated derepression of valyl-tRNA synthetase formation in both of these mutants, as compared with the normal strain. In addition, it was observed that these branched-chain synthetase activities were reduced in steady-state cultures of several ilvA point mutants. However, upon the introduction of the ilv operon to these ilvA mutants by use of lambda bacteriophage, there was a specific increase in the branched-chain synthetase activities to levels comparable to those of the normal strain. These results support our previous findings that the stability and repression control of synthesis of these synthetases require some product(s) missing in the ilvDAC deletion strain and strongly suggest this component is some form of the ilvA gene product, threonine deaminase. PMID:348689

  16. Inactivation and dissociation of S-adenosylmethionine synthetase by modification of sulfhydryl groups and its possible occurrence in cirrhosis.

    PubMed

    Corrales, F; Cabrero, C; Pajares, M A; Ortiz, P; Martin-Duce, A; Mato, J M

    1990-02-01

    Catalytically active human and rat liver S-adenosylmethionine synthetase exists mainly in tetramer and dimer form. In liver biopsy samples from cirrhotic patients a marked reduction in total S-adenosylmethionine synthetase activity and a specific loss of the tetrameric form of the enzyme exist. We have investigated the possible role of sulfhydryl groups in maintaining the structure and activity of S-adenosylmethionine synthetase. Both forms of S-adenosylmethionine synthetase are rapidly inactivated by N-ethylmaleimide, and the loss of enzyme activity correlates with the incorporation of approximately 2 moles N-ethylmaleimide per mole of subunit. In addition, reaction with N-ethylmaleimide resulted in displacement of the tetramer-dimer equilibrium of the enzyme toward the dimer, but no monomer was detected under these conditions. A catalytically active monomeric S-adenosylmethionine synthetase was detected in the cytosolic extract from a liver biopsy sample from a cirrhotic patient, supporting our model for the structure of S-adenosylmethionine synthetase. Because treatment of S-adenosylmethionine synthetase with N-ethylmaleimide resembles the situation of this enzyme in cirrhotic patients, it is proposed that impaired protection of the enzyme from oxidizing agents caused by a decreased synthesis of glutathione can explain the diminished synthesis of S-adenosylmethionine in liver cirrhosis. PMID:2307400

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

    PubMed

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

    1996-11-01

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

  18. Helicobacter pylori Glutamine Synthetase Lacks Features Associated with Transcriptional and Posttranslational Regulation

    PubMed Central

    Garner, Rachel M.; Fulkerson, John; Mobley, Harry L. T.

    1998-01-01

    Helicobacter pylori urease, produced in abundance, is indispensable for the survival of H. pylori in animal hosts. Urea is hydrolyzed by the enzyme, resulting in the liberation of excess ammonia, some of which neutralizes gastric acid. The remaining ammonia is assimilated into protein by glutamine synthetase (EC 6.3.1.2), which catalyzes the reaction: NH3 + glutamate + ATP→glutamine + ADP + Pi. We hypothesized that glutamine synthetase plays an unusually critical role in nitrogen assimilation by H. pylori. We developed a phenotypic screen to isolate genes that contribute to the synthesis of a catalytically active urease. Escherichia coli SE5000 transformed with plasmid pHP808 containing the entire H. pylori urease gene cluster was cotransformed with a pBluescript plasmid library of the H. pylori ATCC 43504 genome. A weakly urease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide sequencing. Among other genes, the gene for glutamine synthetase was identified. The complete 1,443-bp glnA gene predicts a polypeptide of 481 amino acid residues with a molecular weight of 54,317; this was supported by maxicell analysis of cloned glnA expressed in E. coli. The top 10 homologs were all bacterial glutamine synthetases, including Salmonella typhimurium glnA. The ATP-binding motif GDNGSG (residues 272 to 277) of H. pylori GlnA exactly matched and aligned with the sequence in 8 of the 10 homologs. The adenylation site found in the top 10 homologs (consensus sequence, NLYDLP) is replaced in H. pylori by NLFKLT (residues 405 to 410). Since the Tyr (Y) residue is the target of adenylation and since the H. pylori glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within this motif. Cloned H. pylori glnA complemented a glnA mutation in E. coli, and GlnA enzyme activity could be measured spectrophotometrically. In an attempt to produce a GlnA-deficient mutant of H. pylori, a kanamycin resistance cassette was cloned

  19. (Radioiodinated free fatty acids)

    SciTech Connect

    Knapp, Jr., F. F.

    1987-12-11

    The traveler participated in the Second International Workshop on Radioiodinated Free Fatty Acids in Amsterdam, The Netherlands where he presented an invited paper describing the pioneering work at the Oak Ridge National Laboratory (ORNL) involving the design, development and testing of new radioiodinated methyl-branched fatty acids for evaluation of heart disease. He also chaired a technical session on the testing of new agents in various in vitro and in vivo systems. He also visited the Institute for Clinical and Experimental Nuclear Medicine in Bonn, West Germany, to review, discuss, plan and coordinate collaborative investigations with that institution. In addition, he visited the Cyclotron Research Center in Liege, Belgium, to discuss continuing collaborative studies with the Osmium-191/Iridium-191m radionuclide generator system, and to complete manuscripts and plan future studies.

  20. 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

  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. Primary structure of histidine-tRNA synthetase and characterization of hisS transcripts.

    PubMed

    Freedman, R; Gibson, B; Donovan, D; Biemann, K; Eisenbeis, S; Parker, J; Schimmel, P

    1985-08-25

    Histidine-tRNA synthetase is one of the smallest bacterial aminoacyl-tRNA synthetases. It is less than one-half the size of the largest aminoacyl-tRNA synthetases. The entire nucleotide sequence of the Escherichia coli hisS locus was determined. The coding region is comprised of 424 codons, and the sequence was determined for 200 nucleotides on the 5'- and 3'-sides of the coding region. The translated nucleotide sequence was confirmed extensively by independent amino acid sequence information obtained by Edman degradations of purified peptides and by measurements of peptide masses by fast atom bombardment mass spectrometry. A significant sequence alignment of four bacterial aminoacyl-tRNA synthetases was reported recently (Webster, T., Tsai, H., Kula, M., Mackie, G., and Schimmel, P. (1984) Science 226, 1315-1317). Although the four enzymes vary considerably in length, this match occurs within the first 100 amino acids of each of the four enzymes and is in the segment believed to be part of the catalytic core. But no strong alignment could be found of the histidine sequence with these four tRNA synthetase sequences. This enzyme may be derived, therefore, from a different progenitor. Previous work suggested that three places in the hisS 5'-noncoding sequence could be promoter sites for RNA polymerase (Eisenbeis, S. J., and Parker, J. (1982) Gene 18, 107-114). We detected a 1400-nucleotide RNA species by RNA blot analysis with a hisS-specific probe. S1 nuclease mapping demonstrated a 5'-end to the RNA species occurs at -67 +/- 1, relative to the first nucleotide of the coding region. This position coincides with the predicted start site for transcription from one of the previously proposed promoter sites. PMID:2991272

  3. Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species

    PubMed Central

    Anderson, J. W.; Fowden, L.

    1970-01-01

    1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive. PMID:5493504

  4. Transfer of fatty acids from the 1-position of phosphatidyl-ethanolamine to the major outer membrane lipoprotein of E coli

    SciTech Connect

    Jackowski, S.; Rock, C.O.

    1986-05-01

    The fatty acids esterified to Braun's lipoprotein are derived from the phospholipid pool in E. coli. Mutants lacking acyl-CoA synthetase activity (fadD) incorporated extracellular fatty acids specifically into the 1-position of phosphatidylethanolamine (PtdEtn). This pathway was blocked by chloramphenicol and was depressed by preventing the acylation of the amino terminus of the lipoprotein with globomycin. Transfer of fatty acids to lipoprotein was investigated in fadD mutants harboring hybrid plasmids containing either the lipoprotein gene or a lipoprotein-..beta..-lactamase gene fusion under control of the lactose promoter. Labeling of the 1-position of the PtdEtn pool prior to induction of lipoprotein biosynthesis resulted in the transfer of fatty acids from PtdEtn to the lipoproteins. Induction of lipoprotein synthesis in the presence of exogenous (1-/sup 14/C)palmitate increased the amount of radioactivity entering the PtdEtn pool and efficiently labeled lipoprotein acyl moieties. Lipoprotein fatty acids derived from the 1-position of PtdEtn were resistant to hydroxylamine hydrolysis, and globomycin reduced the incorporation of exogenous (1-/sup 14/C)palmitic acid into lipoproteins by 80% suggesting that the fatty acid is attached to the amino terminus. These data illustrate the metabolic relationship between turnover of fatty acids in the 1-position of PtdEtn and the maturation of the major outer membrane lipoprotein.

  5. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  6. Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling

    SciTech Connect

    Pollard, M.; Ohlrogge, J.

    1999-12-01

    Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [{sup 13}C{sub 2}{sup 18}O{sub 2}]Acetate was incubated with spinach (Spinacia oleracea) leaves and the {sup 18}O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectrometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an {sup 18}O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the {sup 18}O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of {sup 18}O or, less likely, complete loss of {sup 18}O, but not a 50% loss of {sup 18}O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of prokaryotic and eukaryotic lipids have both been confirmed.

  7. Characterization of the promoter region of the bovine long-chain acyl-CoA synthetase 1 gene: Roles of E2F1, Sp1, KLF15, and E2F4

    PubMed Central

    Zhao, Zhi-Dong; Zan, Lin-Sen; Li, An-Ning; Cheng, Gong; Li, Shi-Jun; Zhang, Ya-Ran; Wang, Xiao-Yu; Zhang, Ying-Ying

    2016-01-01

    The nutritional value and eating qualities of beef are enhanced when the unsaturated fatty acid content of fat is increased. Long-chain acyl-CoA synthetase 1 (ACSL1) plays key roles in fatty acid transport and degradation, as well as lipid synthesis. It has been identified as a plausible functional and positional candidate gene for manipulations of fatty acid composition in bovine skeletal muscle. In the present study, we determined that bovine ACSL1was highly expressed in subcutaneous adipose tissue and longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ACSL1 regulation, we cloned and characterized the promoter region of ACSL1. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified multiple transcriptional start sites (TSSs) in its promoter region. Using a series of 5′ deletion promoter plasmids in luciferase reporter assays, we found that the proximal minimal promoter of ACSL1 was located within the region −325/−141 relative to the TSS and it was also located in the predicted CpG island. Mutational analysis and electrophoretic mobility shift assays demonstrated that E2F1, Sp1, KLF15 and E2F4 binding to the promoter region drives ACSL1 transcription. Together these interactions integrate and frame a key functional role for ACSL1 in mediating the lipid composition of beef. PMID:26782942

  8. 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

  9. Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

    PubMed Central

    McGinnis, Etheleen; Williams, Ann C.; Williams, L. S.

    1974-01-01

    The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid. PMID:4604302

  10. Nonalcoholic fatty liver disease.

    PubMed

    Brunt, Elizabeth M; Wong, Vincent W-S; Nobili, Valerio; Day, Christopher P; Sookoian, Silvia; Maher, Jacquelyn J; Bugianesi, Elisabetta; Sirlin, Claude B; Neuschwander-Tetri, Brent A; Rinella, Mary E

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a disorder characterized by excess accumulation of fat in hepatocytes (nonalcoholic fatty liver (NAFL)); in up to 40% of individuals, there are additional findings of portal and lobular inflammation and hepatocyte injury (which characterize nonalcoholic steatohepatitis (NASH)). A subset of patients will develop progressive fibrosis, which can progress to cirrhosis. Hepatocellular carcinoma and cardiovascular complications are life-threatening co-morbidities of both NAFL and NASH. NAFLD is closely associated with insulin resistance; obesity and metabolic syndrome are common underlying factors. As a consequence, the prevalence of NAFLD is estimated to be 10-40% in adults worldwide, and it is the most common liver disease in children and adolescents in developed countries. Mechanistic insights into fat accumulation, subsequent hepatocyte injury, the role of the immune system and fibrosis as well as the role of the gut microbiota are unfolding. Furthermore, genetic and epigenetic factors might explain the considerable interindividual variation in disease phenotype, severity and progression. To date, no effective medical interventions exist that completely reverse the disease other than lifestyle changes, dietary alterations and, possibly, bariatric surgery. However, several strategies that target pathophysiological processes such as an oversupply of fatty acids to the liver, cell injury and inflammation are currently under investigation. Diagnosis of NAFLD can be established by imaging, but detection of the lesions of NASH still depend on the gold-standard but invasive liver biopsy. Several non-invasive strategies are being evaluated to replace or complement biopsies, especially for follow-up monitoring. PMID:27188459

  11. Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase.

    PubMed Central

    Sissler, M; Eriani, G; Martin, F; Giegé, R; Florentz, C

    1997-01-01

    Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArgand non-cognate tRNAAspare described. This work was motivated by the fact that the in vitro transcript of tRNAAspis of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAspis recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNAAspis contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArgand tRNAAsp. The mirror image alternative interaction patterns of unmodified tRNAAspwith either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNAAspand either ArgRS and AspRS is solely governed by the proteins. PMID:9396794

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

    DOEpatents

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

    2009-08-18

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

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

    DOEpatents

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

    2009-12-29

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

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

    DOEpatents

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

    2011-10-04

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

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

    DOEpatents

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

    2014-03-11

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

  16. Inhibitory effect of quinolone antimicrobial and nonsteroidal anti-inflammatory drugs on a medium chain acyl-CoA synthetase.

    PubMed

    Kasuya, F; Hiasa, M; Kawai, Y; Igarashi, K; Fukui, M

    2001-08-01

    The inhibitory effects of quinolone antimicrobial agents and nonsteroidal anti-inflammatory drugs on purified mouse liver mitochondrial medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation were examined, using hexanoic acid as a substrate. Enoxacin, ofloxacin, nalidixic acid, diflunisal, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid, which do not act as substrates, were potent inhibitors. Diflunisal, nalidixic acid, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid inhibited competitively this medium chain acyl-CoA synthetase with K(i) values of 0.6, 12.4, 19.6, 13.4, and 15.0 microM, respectively. Enoxacin and ofloxacin inhibited this medium chain acyl-CoA synthetase in a mixed-type manner with K(i) values of 23.7 and 38.2 microM, respectively. Felbinac, which is a substrate, inhibited the activity of this medium chain acyl-CoA synthetase for hexanoic acid (IC50 = 25 microM). The concomitant presence of enoxacin and felbinac strongly inhibited this medium chain acyl-CoA synthetase. These findings indicate that medium chain acyl-CoA synthetases may be influenced by quinolone antimicrobial and nonsteroidal anti-inflammatory drugs. PMID:11434910

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

    PubMed

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman; Ottenwälder, Birgit; Schnölzer, Martina; Kartenbeck, Jürgen; Lyer, Stefan; Autschbach, Frank; Poustka, Annemarie; Otto, Herwart F; Mollenhauer, Jan

    2004-02-01

    Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine. PMID:14743501

  18. Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane.

    PubMed Central

    Mannaerts, G P; Van Veldhoven, P; Van Broekhoven, A; Vandebroek, G; Debeer, L J

    1982-01-01

    1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes. PMID:7115321

  19. Treatment of Fatty Acid Oxidation Disorders

    MedlinePlus

    ... of fatty acid oxidation disorders Treatment of fatty acid oxidation disorders E-mail to a friend Please ... page It's been added to your dashboard . Fatty acid oxidation disorders are rare health conditions that affect ...

  20. 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

  1. Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase.

    PubMed Central

    Walter, P; Gangloff, J; Bonnet, J; Boulanger, Y; Ebel, J P; Fasiolo, F

    1983-01-01

    The sequence of a 5-kilobase DNA insert containing the structural gene for yeast cytoplasmic methionyl-tRNA synthetase has been determined and a unique open reading frame of 2,253 nucleotides encoding a polypeptide chain of 751 amino acids (Mr, 85,500) has been characterized. The data obtained on the purified enzyme (subunit size, amino acid composition, and COOH-terminal sequence) are consistent with the gene structure. The protein sequence deduced from the nucleotide sequence reveals no obvious internal repeats. This protein sequence shows a high degree of homology with that of Escherichia coli methionyl-tRNA synthetase within a region that forms the putative methionyl adenylate binding site. This strongly suggests that both proteins derive from a common ancestor. PMID:6341994

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

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

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

    PubMed

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

    2014-02-01

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

  5. Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase

    PubMed Central

    Keller, Tracy L.; Zocco, Davide; Sundrud, Mark S.; Hendrick, Margaret; Edenius, Maja; Yum, Jina; Kim, Yeon-Jin; Lee, Hak-kyo; Cortese, Joseph F.; Wirth, Dyann; Dignam, John David; Rao, Anjana; Yeo, Chang-Yeol; Mazitschek, Ralph; Whitman, Malcolm

    2011-01-01

    Febrifugine, one of the fifty fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity whilst its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis, and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of Th17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response pathway (AAR). Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS) inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural products. This work both explains the molecular mechanism of a promising family of therapeutics, and highlights the AAR pathway as an important drug target for promoting inflammatory resolution. PMID:22327401

  6. Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

    PubMed

    Hasegawa, T; Yokogawa, T

    2000-01-01

    A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase. PMID:12903242

  7. Recoding Aminoacyl-tRNA Synthetases for Synthetic Biology by Rational Protein-RNA Engineering

    PubMed Central

    2015-01-01

    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 tRNAGln by over 105-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

  8. Dual inhibitory effects of dimethyl sulfoxide on poly(ADP-ribose) synthetase.

    PubMed

    Banasik, M; Ueda, K

    1999-01-01

    Dimethyl sulfoxide (DMSO), a solvent popularly used for dissolving water-insoluble compounds, is a weak inhibitor of poly(ADP-ribose) synthetase, that is a nuclear enzyme producing (ADP-ribose)n from NAD+. The inhibitory mode and potency depend on the concentration of substrate, NAD+, as well as the temperature of the reaction; at micromolar concentrations of NAD+, the inhibition by DMSO is biphasic at 37 degrees C, but is monophasic and apparently competitive with NAD+ at 25 degrees C. DMSO, on the other hand, diminishes dose-dependently and markedly the inhibitory potency of benzamide and other inhibitors. Other organic solvents, ethanol and methanol, also show a biphasic effect on the synthetase activity at different concentrations. PMID:10445046

  9. S-adenosyl-L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis.

    PubMed

    Duce, A M; Ortíz, P; Cabrero, C; Mato, J M

    1988-01-01

    We have measured the activity S-adenosyl-L-methionine synthetase in liver biopsies from a group of controls (n = 17) and in 26 cirrhotics (12 alcoholic and 14 posthepatic). The activity of this enzyme was markedly reduced in the group of cirrhotics (285 +/- 32 pmoles per min per mg protein) when compared with that observed in controls (505 +/- 37 pmoles per min per mg protein). No differences in S-adenosyl-L-methionine synthetase was observed between both groups of cirrhotics. Similarly, a marked reduction in the activity phospholipid methyltransferase was also observed in liver biopsies from the same group of cirrhotics (105 +/- 12 pmoles per min per mg protein) when compared with the control subjects (241 +/- 13 pmoles per min per mg protein). Again, no difference in the activity of this enzyme was observed between both groups of cirrhotics. These results indicated a marked deficiency in the metabolism of S-adenosyl-L-methionine in cirrhosis. PMID:3338721

  10. Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation.

    PubMed Central

    Francklyn, Christopher; Perona, John J; Puetz, Joern; Hou, Ya-Ming

    2002-01-01

    Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code. PMID:12458790

  11. Effects of prostaglandins and prostaglandin synthetase inhibitors on acutely obstructed kidneys in the dog.

    PubMed

    Zwergel, U; Zwergel, T; Ziegler, M

    1991-01-01

    An intact canine model was developed to study the effects of prostaglandins (PG) and prostaglandin synthetase inhibitors on acutely obstructed kidneys. Totally implanted nephrostomy tubes were placed to measure renal pelvic pressure. Complete ureteral obstruction was obtained with a Fogarty balloon catheter inflated in the distal ureter; by this method renal pelvic pressure reached 40-50 mm Hg. Renal pelvic pressure was reduced after intravenous indomethacin and dipyrone administration, whereas blood pressure showed no major changes. Exogenous prostaglandins had both immediate and contrary effects: PGE2 caused a significant decrease, whereas PGF2 alpha caused a significant increase in renal pelvic and blood pressure. The reduced rise in renal pelvic pressure appears to be the main reason for the analgesic effects of prostaglandin synthetase inhibitors. The efficiency of these drugs in the treatment of renal colic is supported by this study, that of prostaglandins cannot be proved. PMID:1792708

  12. Structure of Human Phosphopantothenoylcysteine Synthetase at 2.3 Å Resolution

    SciTech Connect

    Manoj, N.; Strauss, E.; Begley, T.P.; Ealick, S.E.

    2010-12-01

    The structure of human phosphopantothenoylcysteine (PPC) synthetase was determined at 2.3 {angstrom} resolution. PPC synthetase is a dimer with identical monomers. Some features of the monomer fold resemble a group of NAD-dependent enzymes, while other features resemble the ribokinase fold. The ATP, phosphopantothenate, and cysteine binding sites were deduced from modeling studies. Highly conserved ATP binding residues include Gly43, Ser61, Gly63, Gly66, Phe230, and Asn258. Highly conserved phosphopantothenate binding residues include Asn59, Ala179, Ala180, and Asp183 from one monomer and Arg55 from the adjacent monomer. The structure predicts a ping pong mechanism with initial formation of an acyladenylate intermediate, followed by release of pyrophosphate and attack by cysteine to form the final products PPC and AMP.

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

    PubMed

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

    2006-09-01

    Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms. PMID:16772660

  14. Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

    PubMed Central

    Hughes, Julia; Mellows, Graham

    1978-01-01

    The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RCrel, whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNAIle. Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first. PMID:365175

  15. 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

  16. Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

    1990-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  18. A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

    PubMed Central

    Lloyd, A J; Thomann, H U; Ibba, M; Söll, D

    1995-01-01

    We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS. PMID:7659511

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

    PubMed Central

    Pérez-Delgado, Carmen M.; García-Calderón, Margarita; Márquez, Antonio J.; Betti, Marco

    2015-01-01

    It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4+ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity. PMID:26091523

  20. Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene.

    PubMed Central

    Luo, D; Leautey, J; Grunberg-Manago, M; Putzer, H

    1997-01-01

    We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749. The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli. Extracts of B. subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity. Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream. The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination. We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr). Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion. Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon. Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes. PMID:9098041

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

    PubMed Central

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

    2009-01-01

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

  2. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

    PubMed

    de Jong, Bouke Wim; Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2015-03-01

    Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae. PMID:25422103

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

    PubMed

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

    2016-05-01

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

  4. Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa.

    PubMed

    Reimmann, C; Serino, L; Beyeler, M; Haas, D

    1998-11-01

    The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pchEF, encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE (156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensation/cyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchE'-'lacZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led to Fur (ferric uptake regulator)-dependent repression of the pchE'-'lacZ fusion. A translational pchD'-'lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa. PMID:9846750

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

    PubMed

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

    2000-10-24

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

  6. Food safety: Structure and expression of the asparagine synthetase gene family of wheat

    PubMed Central

    Gao, Runhong; Curtis, Tanya Y.; Powers, Stephen J.; Xu, Hongwei; Huang, Jianhua; Halford, Nigel G.

    2016-01-01

    Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat. PMID:27110058

  7. Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

    PubMed Central

    Felden, Brice; Giegé, Richard

    1998-01-01

    Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions. PMID:9724720

  8. Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase.

    PubMed

    Felden, B; Giegé, R

    1998-09-01

    Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3'-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2-3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions. PMID:9724720

  9. Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase.

    PubMed Central

    Eriani, G; Dirheimer, G; Gangloff, J

    1989-01-01

    The gene coding for Escherichia coli arginyl-tRNA synthetase (argS) was isolated as a fragment of 2.4 kb after analysis and subcloning of recombinant plasmids from the Clarke and Carbon library. The clone bearing the gene overproduces arginyl-tRNA synthetase by a factor 100. This means that the enzyme represents more than 20% of the cellular total protein content. Sequencing revealed that the fragment contains a unique open reading frame of 1734 bp flanked at its 5' and 3' ends respectively by 247 bp and 397 bp. The length of the corresponding protein (577 aa) is well consistent with earlier Mr determination (about 70 kd). Primer extension analysis of the ArgRS mRNA by reverse transcriptase, located its 5' end respectively at 8 and 30 nucleotides downstream of a TATA and a TTGAC like element (CTGAC) and 60 nucleotides upstream of the unusual translation initiation codon GUG; nuclease S1 analysis located the 3'-end at 48 bp downstream of the translation termination codon. argS has a codon usage pattern typical for highly expressed E. coli genes. With the exception of the presence of a HVGH sequence similar to the HIGH consensus element, ArgRS has no relevant sequence homologies with other aminoacyl-tRNA synthetases. Images PMID:2668891

  10. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

    PubMed Central

    Arnez, J G; Harris, D C; Mitschler, A; Rees, B; Francklyn, C S; Moras, D

    1995-01-01

    The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Images PMID:7556055

  11. Alternative splicing creates two new architectures for human tyrosyl-tRNA synthetase

    PubMed Central

    Wei, Zhiyi; Xu, Zhiwen; Liu, Xiaotian; Lo, Wing-Sze; Ye, Fei; Lau, Ching-Fun; Wang, Feng; Zhou, Jie J.; Nangle, Leslie A.; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

    2016-01-01

    Many human tRNA synthetases evolved alternative functions outside of protein synthesis. These functions are associated with over 200 splice variants (SVs), most of which are catalytic nulls that engender new biology. While known to regulate non-translational activities, little is known about structures resulting from natural internal ablations of any protein. Here, we report analysis of two closely related, internally deleted, SVs of homodimeric human tyrosyl-tRNA synthetase (TyrRS). In spite of both variants ablating a portion of the catalytic core and dimer-interface contacts of native TyrRS, each folded into a distinct stable structure. Biochemical and nuclear magnetic resonance (NMR) analysis showed that the internal deletion of TyrRSΔE2–4 SV gave an alternative, neomorphic dimer interface ‘orthogonal’ to that of native TyrRS. In contrast, the internal C-terminal splice site of TyrRSΔE2–3 prevented either dimerization interface from forming, and yielded a predominantly monomeric protein. Unlike ubiquitous TyrRS, the neomorphs showed clear tissue preferences, which were distinct from each other. The results demonstrate a sophisticated structural plasticity of a human tRNA synthetase for architectural reorganizations that are preferentially elicited in specific tissues. PMID:26773056

  12. Localization of the L-glutamine synthetase gene to chromosome 1q23.

    PubMed

    Clancy, K P; Berger, R; Cox, M; Bleskan, J; Walton, K A; Hart, I; Patterson, D

    1996-12-15

    Glutamine synthetase (E.C. 6.3.1.2) is expressed throughout the body and plays an important role in controlling body pH and in removing ammonia from the circulation. The enzyme clears L-glutamate, the major neurotransmitter in the central nervous system, from neuronal synapses. The enzyme is a very sensitive marker of many disease and aging processes, especially those involving reactive oxygen species. This report describes the localization of the enzyme to chromosome 1 by PCR analysis of a human/rodent somatic cell hybrid panel. We also describe the localization of a recently described pseudogene to chromosome 9. Further localization of the glutamine synthetase gene locus to 1q23 was accomplished by fluorescence in situ hybridization. The glutamine synthetase gene was mapped to five CEPH megaYACs between the polymorphic PCR markers D1S117 and D1S466 by analysis of the Whitehead Institute's recently described chromosome 1 contig map. PMID:8975719

  13. The structure of tryptophanyl-tRNA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs

    PubMed Central

    Arakaki, Tracy L; Carter, Megan; Napuli, Alberto J; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2010-01-01

    The 2.1 Å crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue α-helix. This helix replaces a β-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this β-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (α2)2 homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal α-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS. PMID:20438846

  14. Mutants of Salmonella typhimurium with an Altered Leucyl-Transfer Ribonucleic Acid Synthetase

    PubMed Central

    Alexander, Renee R.; Calvo, J. M.; Freundlich, M.

    1971-01-01

    Two trifluoroleucine-resistant mutants of Salmonella typhimurium, strains CV69 and CV117, had an altered leucyl-transfer ribonucleic acid (tRNA) synthetase. The mutant enzymes had higher apparent Km values for leucine (ca. 10-fold) and lower specific activities (ca. twofold) than the parent enzyme when tested in crude extracts. Preparations of synthetase purified ca. 60-fold from the parent and strain CV117 differed sixfold in their leucine Km values. In addition, the mutant enzyme was inactivated faster than the parent enzyme at 50 C. The growth rates of strains CV69 and CV117 at 37 C were not significantly different from that of the parent, whereas at 42 C strain CV69 grew more slowly than the parent. Leucine-, valine-, and isoleucine-forming enzymes were partially derepressed when the mutants were grown in minimal medium; the addition of leucine repressed these enzymes to wild-type levels. During growth in minimal medium, the proportion of leucine tRNA that was charged in the mutants was about 75% of that in the parent. The properties of strain CV117 were shown to result from a single mutation located near gal at minute 18 on the genetic map. These studies suggest that leucyl-tRNA synthetase is involved in repression of the enzymes required for the synthesis of branched-chain amino acids. PMID:4928008

  15. Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis.

    PubMed Central

    Vander Horn, P B; Zahler, S A

    1992-01-01

    The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced. A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles. The leuS1 mutation was mapped to 270 degrees on the chromosome. Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon. The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B. subtilis ilv-leu operon. The DNA of the B. subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E. coli. PMID:1317842

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

    PubMed

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

    2016-10-01

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

  17. Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

    PubMed Central

    Ranade, Ranae M.; Zhang, Zhongsheng; Dranow, David M.; Myers, Janette B.; Choi, Ryan; Nakazawa Hewitt, Steve; Edwards, Thomas E.; Davies, Douglas R.; Lorimer, Donald; Boyle, Stephen M.; Barrett, Lynn K.; Buckner, Frederick S.; Fan, Erkang; Van Voorhis, Wesley C.

    2016-01-01

    We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment. PMID:27500735

  18. CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria.

    PubMed

    Klemke, Friederike; Nürnberg, Dennis J; Ziegler, Karl; Beyer, Gabriele; Kahmann, Uwe; Lockau, Wolfgang; Volkmer, Thomas

    2016-03-01

    Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide β-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant. PMID:26781249

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

    SciTech Connect

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

    1987-04-01

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

  20. Examination of the mechanism of sucrose synthetase by positional isotope exchange

    SciTech Connect

    Singh, A.N.; Hester, L.S.; Raushel, F.M.

    1987-02-25

    The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. (beta-/sup 18/O/sub 2/, alpha beta-/sup 18/O)UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of (beta-/sup 18/O/sub 2/, alpha beta-/sup 18/O)UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.

  1. Alternative splicing creates two new architectures for human tyrosyl-tRNA synthetase.

    PubMed

    Wei, Zhiyi; Xu, Zhiwen; Liu, Xiaotian; Lo, Wing-Sze; Ye, Fei; Lau, Ching-Fun; Wang, Feng; Zhou, Jie J; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

    2016-02-18

    Many human tRNA synthetases evolved alternative functions outside of protein synthesis. These functions are associated with over 200 splice variants (SVs), most of which are catalytic nulls that engender new biology. While known to regulate non-translational activities, little is known about structures resulting from natural internal ablations of any protein. Here, we report analysis of two closely related, internally deleted, SVs of homodimeric human tyrosyl-tRNA synthetase (TyrRS). In spite of both variants ablating a portion of the catalytic core and dimer-interface contacts of native TyrRS, each folded into a distinct stable structure. Biochemical and nuclear magnetic resonance (NMR) analysis showed that the internal deletion of TyrRSΔE2-4 SV gave an alternative, neomorphic dimer interface 'orthogonal' to that of native TyrRS. In contrast, the internal C-terminal splice site of TyrRSΔE2-3 prevented either dimerization interface from forming, and yielded a predominantly monomeric protein. Unlike ubiquitous TyrRS, the neomorphs showed clear tissue preferences, which were distinct from each other. The results demonstrate a sophisticated structural plasticity of a human tRNA synthetase for architectural reorganizations that are preferentially elicited in specific tissues. PMID:26773056

  2. Dual targeting is the rule for organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana

    PubMed Central

    Duchêne, Anne-Marie; Giritch, Anatoli; Hoffmann, Beate; Cognat, Valérie; Lancelin, Dominique; Peeters, Nemo M.; Zaepfel, Marlyse; Maréchal-Drouard, Laurence; Small, Ian D.

    2005-01-01

    In plants, protein synthesis occurs in the cytosol, mitochondria, and plastids. Each compartment requires a full set of tRNAs and aminoacyl-tRNA synthetases. We have undertaken a systematic analysis of the targeting of organellar aminoacyl-tRNA synthetases in the model plant Arabidopsis thaliana. Dual targeting appeared to be a general rule. Among the 24 identified organellar aminoacyl-tRNA synthetases (aaRSs), 15 (and probably 17) are shared between mitochondria and plastids, and 5 are shared between cytosol and mitochondria (one of these aaRSs being present also in chloroplasts). Only two were shown to be uniquely chloroplastic and none to be uniquely mitochondrial. Moreover, there are no examples where the three aaRS genes originating from the three ancestral genomes still coexist. These results indicate that extensive exchange of aaRSs has occurred during evolution and that many are now shared between two or even three compartments. The findings have important implications for studies of the translation machinery in plants and on protein targeting and gene transfer in general. PMID:16251277

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

    PubMed

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

    2016-08-10

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

  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. Isolation and Partial Characterization of Temperature-Sensitive Escherichia coli Mutants with Altered Leucyl- and Seryl-Transfer Ribonucleic Acid Synthetases

    PubMed Central

    Low, B.; Gates, F.; Goldstein, T.; Söll, D.

    1971-01-01

    Two temperature-sensitive mutants of Escherichia coli have been found in which the conditional growth is a result of a thermosensitive leucyl-transfer ribonucleic acid (tRNA) synthetase and seryl-tRNA synthetase, respectively. The corresponding genetic loci, leuS and serS, cotransduce with lip and serC, respectively. As a result of the mutationally altered leucyl-tRNA synthetase, some leucine-, valine-, and isoleucine-forming enzymes were derepressed. Thus, leucyl-tRNA synthetase is involved in the repression of the enzymes needed for the synthesis of branched-chain amino acids. PMID:4942762

  6. 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

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

    PubMed

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

    2015-07-01

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

  8. Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

    PubMed

    Brown, J R; Doolittle, W F

    1995-03-28

    Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an

  9. Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates†

    PubMed Central

    Fontes, Rui; Günther Sillero, Maria A.; Sillero, Antonio

    1998-01-01

    Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p4A) and adenosine 5′-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), and adenosine(5′)pentaphospho(5′)adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The Km values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates). PMID:9620965

  10. Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

    PubMed Central

    Cheesbrough, Thomas M.

    1989-01-01

    Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not. PMID:16666840

  11. Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances.

    PubMed

    Lee, Je Min; Lee, Hyungjae; Kang, SeokBeom; Park, Woo Jung

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism. PMID:26742061

  12. Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances

    PubMed Central

    Lee, Je Min; Lee, Hyungjae; Kang, SeokBeom; Park, Woo Jung

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism. PMID:26742061

  13. Abiotic synthesis of fatty acids

    NASA Technical Reports Server (NTRS)

    Leach, W. W.; Nooner, D. W.; Oro, J.

    1978-01-01

    The formation of fatty acids by Fischer-Tropsch-type synthesis was investigated with ferric oxide, ammonium carbonate, potassium carbonate, powdered Pueblito de Allende carbonaceous chondrite, and filings from the Canyon Diablo meteorite used as catalysts. Products were separated and identified by gas chromatography and mass spectrometry. Iron oxide, Pueblito de Allende chondrite, and Canyon Diablo filings in an oxidized catalyst form yielded no fatty acids. Canyon Diablo filings heated overnight at 500 C while undergoing slow purging by deuterium produced fatty acids only when potassium carbonate was admixed; potassium carbonate alone also produced these compounds. The active catalytic combinations gave relatively high yields of aliphatic and aromatic hydrocarbons; substantial amounts of n-alkenes were almost invariably observed when fatty acids were produced; the latter were in the range C6 to C18, with maximum yield in C9 or 10.

  14. Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

    PubMed Central

    Sellami, M; Rether, B; Gangloff, J; Ebel, J P; Bonnet, J

    1983-01-01

    We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs. Images PMID:6344009

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

    PubMed

    Brattsten, L B; Wilkinson, C F

    1975-07-01

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

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

    PubMed Central

    Brattsten, L B; Wilkinson, C F

    1975-01-01

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

  17. Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae.

    PubMed

    Flores Martinez, A; Schwencke, J

    1988-12-22

    The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae. PMID:2974729

  18. Understanding species-specific differences in substrate recognition by Escherichia coli and human prolyl-tRNA synthetases.

    PubMed

    Musier-Forsyth, K; Stehlin, C; Burke, B; Liu, H

    1997-01-01

    Class II human prolyl-tRNA synthetase (ProRS) aminoacylates in vitro transcribed human tRNA(Pro) with kinetic parameters that are similar to those previously determined for aminoacylation of Escherichia coli tRNA(Pro) by its cognate synthetase. As in the bacterial system, large decreases in aminoacylation by human ProRS occur upon mutating anticodon positions G35 and G36 of human tRNA(Pro). The N73 'discriminator' base and the first and third base pairs of the acceptor stem vary between the E.coli and human isoacceptor groups. In contrast to the E. coli synthetase, the human enzyme does not appear to recognize these elements, since mutations at these positions do not significantly affect cognate synthetase charging. E. coli ProRS does not cross-aminoacylate human tRNA(Pro), and the bacterial tRNA(Pro) is a poor substrate for the human enzyme. Mutations in both the tRNAs and the synthetases have been made in an effort to identify elements in each system responsible for blocking cross-species aminoacylation. Alignment of all known ProRS primary sequences from different species reveals particularly low overall sequence homology, as well as two distinct groups of enzymes. The sequence divergence between E. coli and human ProRSs helps to explain the species-specific differences in the RNA code for aminoacylation of tRNA(Pro). PMID:9478190

  19. MASS SPECTROMETRY OF FATTY ALDEHYDES

    PubMed Central

    Berdyshev, Evgeny V.

    2011-01-01

    Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. PMID:21930240

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

    EPA Science Inventory

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

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

    SciTech Connect

    Chu, Wenchy; Horowitz, J. )

    1991-02-12

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

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

    PubMed

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

    2012-05-01

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

  3. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

    SciTech Connect

    Reger,A.; Carney, J.; Gulick, A.

    2007-01-01

    The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

  4. 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

  5. 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. PMID:11123948

  6. Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans▿

    PubMed Central

    Meng, Yu; Ingram-Smith, Cheryl; Cooper, Leroy L.; Smith, Kerry S.

    2010-01-01

    Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme. PMID:20851904

  7. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  8. Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase.

    PubMed

    Ingram-Smith, Cheryl; Woods, Barrett I; Smith, Kerry S

    2006-09-26

    AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site. PMID:16981708

  9. 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

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

    PubMed

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

    2015-11-10

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

  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. Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

    PubMed Central

    Hoff-Risseti, Caroline; Dörr, Felipe Augusto; Schaker, Patricia Dayane Carvalho; Pinto, Ernani; Werner, Vera Regina; Fiore, Marli Fatima

    2013-01-01

    The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins. PMID:24015317

  13. Control of Escherichia coli lysyl-tRNA synthetase expression by anaerobiosis.

    PubMed Central

    Lévêque, F; Gazeau, M; Fromant, M; Blanquet, S; Plateau, P

    1991-01-01

    Escherichia coli lysyl-tRNA synthetase was previously shown to occur as two distinct species encoded by either the lysS or the lysU gene. The expression of one of these genes, lysU, is under the control of cell growth conditions. To study the regulation of lysU, delta lysS strains were constructed. During aerobic growth at 37 degrees C or below, the amount of the lysU product in the cell is so reduced that delta lysS bacteria grow only poorly. The reduced expression of lysU is not related to the steady-state lysyl-tRNA synthetase concentration in the cell, since the expression of a lysU::lacZ fusion is insensitive to the absence of either lysS or lysU or to the addition of a multi-copy plasmid carrying either lysU or lysS. During anaerobic growth in rich medium, the lysU gene becomes strongly expressed and, in cell extracts, the amount of lysyl-tRNA synthetase activity originating from lysU may become seven times greater than the activity originating from lysS. In minimal medium, lysU expression is only slightly induced. Evidence that the sensitivity of lysU expression to anaerobiosis, as well as to low external pH conditions (E. W. Hickey and I. N. Hirshfield, Appl. Environ. Microbiol. 56:1038-1045, 1990), is governed at the level of transcription is provided. Images FIG. 4 PMID:1744045

  14. 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. PMID:27226617

  15. Novel Insights into Seed Fatty Acid Synthesis and Modification Pathways from Genetic Diversity and Quantitative Trait Loci Analysis of the Brassica C Genome1[OA

    PubMed Central

    Barker, Guy C.; Larson, Tony R.; Graham, Ian A.; Lynn, James R.; King, Graham J.

    2007-01-01

    Natural genetic variation in fatty acid synthesis and modification pathways determine the composition of vegetable oils, which are major components of human diet and renewable products. Based on known pathways we combined diversity and genetic analysis of metabolites to infer the existence of enzymes encoded by distinct loci, and associated these with specific elongation steps or subpathways. A total of 107 lines representing different Brassica genepools revealed considerable variation for 18 seed fatty acid products. The effect of genetic variation within a single biochemical step on subsequent products was demonstrated using a correlation matrix of scatterplots, and by calculating relative step yields. Surprisingly, diploid Brassica oleracea segregating populations had a similar range of variation for individual fatty acids as across the whole genepool. This allowed identification of 22 quantitative trait loci (QTL) associated with activity in the plastid, early stages of synthesis, desaturation, and elongases. Four QTL were assigned to early stages of synthesis, seven to subpathway specific or general elongase activity, one to ketoacyl acyl-carrier protein synthetase, and two each to fatty acid desaturase and either desaturase or fatty acyl-carrier protein thioesterase. An additional 10 QTL had distinct effects but were not assigned specific functions. Where contrasting behavior in more than one subpathway was detected, we inferred QTL specificity for particular combinations of substrate and product. The assignment of enzyme function to QTL was consistent with the known position of some Brassicaeae candidate genes and collinear regions of the Arabidopsis (Arabidopsis thaliana) genome. PMID:17573542

  16. Glutamine synthetase in ribulose 1,5-bisphosphate carboxylase/oxygenase deficient tobacco mutants in cell suspension culture.

    PubMed

    Hirel, B; Nato, A; Martin, F

    1984-06-01

    In two tobacco mutants lacking ribulose, 1,5-bisphosphate carboxylase/oxygenase the amount of glutamine synthetase and its activity were determined and compared with the wild type green cells. It was shown that in these two mutants glutamine synthetase protein content was six times lower than in the wild type. This situation was comparable to that found in etiolated cells where ribulose 1,5-bisphosphate carboxylase/oxygenase was absent. These observations suggest that a common regulatory mechanism might control the dual light dependent biosynthesis of both enzymes. The results have also implications concerning the efficiency of the reassimilation of ammonia by chloroplastic glutamine synthetase during the photorespiratory process. PMID:24253436

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

    PubMed

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

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM. PMID:27125317

  18. Alterations in Dihydropteroate Synthetase in Cell-Free Extracts of Sulfanilamide-Resistant Neisseria meningitidis and Neisseria gonorrhoeae

    PubMed Central

    Ho, Richard I.; Corman, Leonard; Morse, Stephen A.; Artenstein, Malcolm S.

    1974-01-01

    Extracts from Neisseria meningitidis and N. gonorrhoeae with varying susceptibility to sulfanilamide have been investigated for dihydropteroate synthetase activity. Sulfanilamide was a competitive inhibitor of dihydropteroate synthetase with respect to p-aminobenzoate in extracts from both species. Though the Km for p-aminobenzoate was unaffected, the Ki for sulfanilamide increased and the Vmax decreased as the strains' resistance to sulfanilamide increased. Temperature studies have revealed differences in the dihydropteroate synthetase from N. meningitidis and N. gonorrhoeae. A direct relationship was observed between the minimal inhibitory concentration of sulfanilamide determined in vitro and the ratio of Ki/Km. This ratio may be a molecular explanation of sulfanilamide resistance for both N. meningitidis and N. gonorrhoeae. PMID:15825393

  19. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.

    PubMed

    Jeitner, Thomas M; Cooper, Arthur J L

    2014-12-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans. PMID:24136581

  20. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine – relevance to the treatment of neurological diseases

    PubMed Central

    Jeitner, Thomas M.; Cooper, Arthur J. L.

    2013-01-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase with MSO is shown to be biphasic – an initial reversible competitive inhibition (Ki 1.19 mM) is followed by rapid irreversible inactivation. This Ki value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans. PMID:24136581

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

    SciTech Connect

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-02-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

  2. Hydrogen peroxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase.

    PubMed

    Morse, D E; Duncan, H; Hooker, N; Morse, A

    1977-04-15

    Addition of hydrogen peroxide to seawater causes synchronous spawning in gravid male and female abalones, and certain other mollusks as well. This effect is blocked by exposure of the animals to aspirin, an inhibitor of the enzyme catalyzing oxidative synthesis of prostaglandin endoperoxide. Hydrogen peroxide activates this enzymatic reaction in cell-free extracts prepared from abalone eggs (a very rich source of the prostaglandin endoperoxide synthetase); this effect appears to reveal a fundamental property of prostaglandin endoperoxide synthesis. Applicability of these findings to both mariculture and medical purposes is suggested. PMID:403609

  3. Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

    PubMed Central

    Neuberger, Albert; Sandy, John D.; Tait, George H.

    1973-01-01

    1. The `initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the `maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80–90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65–75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the `low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the `high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH

  4. Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans

    PubMed Central

    McGill, Mitchell R.; Cao, Mengde; Svetlov, Archie; Sharpe, Matthew R.; Williams, C. David; Curry, Steven C.; Farhood, Anwar; Jaeschke, Hartmut; Svetlov, Stanislav I.

    2014-01-01

    Context New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. Objective Characterize ASS in APAP hepatotoxicity. Methods ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. Results In mice, ASS increased before injury, peaked before ALT, and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. Conclusions : ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity. PMID:24597531

  5. 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

  6. Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves 1

    PubMed Central

    Yamaya, Tomoyuki; Hayakawa, Toshihiko; Tanasawa, Keisuke; Kamachi, Kazunari; Mae, Tadahiko; Ojima, Kunihiko

    1992-01-01

    To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the

  7. Concentrations of long-chain acyl-acyl carrier proteins during fatty acid synthesis by chloroplasts isolated from pea (Pisum sativum), safflower (Carthamus tinctoris), and amaranthus (Amaranthus lividus) leaves

    SciTech Connect

    Roughan, G.; Nishida, I. )

    1990-01-01

    Fatty acid synthesis from (1-14C)acetate by chloroplasts isolated from peas and amaranthus was linear for at least 15 min, whereas incorporation of the tracer into long-chain acyl-acyl carrier protein (ACP) did not increase after 2-3 min. When reactions were transferred to the dark after 3-5 min, long-chain acyl-ACPs lost about 90% of their radioactivity and total fatty acids retained all of theirs. Half-lives of the long-chain acyl-ACPs were estimated to be 10-15 s. Concentrations of palmitoyl-, stearoyl-, and oleoyl-ACP as indicated by equilibrium labeling during steady-state fatty acid synthesis, ranged from 0.6-1.1, 0.2-0.7, and 0.4-1.6 microM, respectively, for peas and from 1.6-1.9, 1.3-2.6, and 0.6-1.4 microM, respectively, for amaranthus. These values are based on a chloroplast volume of 47 microliters/mg chlorophyll and varied according to the mode of the incubation. A slow increase in activity of the fatty acid synthetase in safflower chloroplasts resulted in long-chain acyl-ACPs continuing to incorporate labeled acetate for 10 min. Upon re-illumination following a dark break, however, both fatty acid synthetase activity and acyl-ACP concentrations increased very rapidly. Palmitoyl-ACP was present at concentrations up to 2.5 microM in safflower chloroplasts, whereas those of stearoyl- and oleoyl-ACPs were in the lower ranges measured for peas. Acyl-ACPs were routinely separated from extracts of chloroplasts that had been synthesising long-chain fatty acids from labeled acetate by a minor modification of the method of Mancha et al. The results compared favorably with those obtained using alternative analytical methods such as adsorption to filter paper and partition chromatography on silicic acid columns.

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

    SciTech Connect

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

    2014-04-01

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

  9. Molecular recognition of proline tRNA by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

    PubMed

    Yokozawa, Junji; Okamoto, Koji; Kawarabayasi, Yutaka; Kuno, Atsushi; Hasegawa, Tsunemi

    2003-01-01

    To investigate the recognition mechanism of tRNA(Pro) by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1, various tRNA(Pro) transcripts were prepared by in vitro transcription system. These transcripts were aminoacylated with proline by overexpressed A. pernix prolyl-tRNA synthetase. From prolylation experiments, recognition elements of A. pernix tRNA(Pro) were determined to be G35 and G36 of anticodon, discriminator base A73, and G1-C72 base pair at acceptor stem end. PMID:14510473

  10. 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.

  11. The nucleotide sequence of the promoter region of hisS, the structural gene for histidyl-tRNA synthetase.

    PubMed

    Eisenbeis, S J; Parker, J

    1982-05-01

    A plasmid has been constructed which carries hisS, the structural gene for histidyl-RNA synthetase of E. coli, on a 1.6-kb fragment bounded by PvuII and BstEII sites. The DNA sequence of both ends of this fragment was determined. The amino-terminal sequence of histidyl-tRNA synthetase was also determined to locate the promoter proximal coding region and the frame in which it is read. Three promoters were identified by consensus criteria. The region surrounding these promoters contains extensive twofold symmetry. PMID:6290315

  12. 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.

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

    PubMed

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

    2003-08-15

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

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

    PubMed

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

    2016-03-01

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

  15. Polyunsaturated Fatty Acids in Children

    PubMed Central

    2013-01-01

    Polyunsaturated fatty acids (PUFAs) are the major components of brain and retina, and are the essential fatty acids with important physiologically active functions. Thus, PUFAs should be provided to children, and are very important in the brain growth and development for fetuses, newborn infants, and children. Omega-3 fatty acids decrease coronary artery disease and improve blood flow. PUFAs have been known to have anti-inflammatory action and improved the chronic inflammation such as auto-immune diseases or degenerative neurologic diseases. PUFAs are used for metabolic syndrome related with obesity or diabetes. However, there are several considerations related with intake of PUFAs. Obsession with the intake of unsaturated fatty acids could bring about the shortage of essential fatty acids that are crucial for our body, weaken the immune system, and increase the risk of heart disease, arrhythmia, and stroke. In this review, we discuss types, physiologic mechanism of action of PUFAs, intake of PUFAs for children, recommended intake of PUFAs, and considerations for the intake of PUFAs. PMID:24224148

  16. Fatty alcohols production by oleaginous yeast.

    PubMed

    Fillet, Sandy; Gibert, Jordi; Suárez, Beatriz; Lara, Armando; Ronchel, Carmen; Adrio, José L

    2015-11-01

    We have engineered Rhodosporidium toruloides to produce fatty alcohols by expressing a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8. Production of fatty alcohols in flasks was achieved in different fermentation media at titers ranging from 0.2 to 2 g/L. In many of the conditions tested, more than 80 % of fatty alcohols were secreted into the cultivation broth. Through fed-batch fermentation in 7 L bioreactors, over 8 g/L of C(16)-C(18) fatty alcohols were produced using sucrose as the substrate. This is the highest titer ever reported on microbial production of fatty alcohols to date. PMID:26318028

  17. Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

    PubMed

    Yamauchi, M; Yamauchi, N; Meuth, M

    1990-07-01

    Successive rounds of chromosome-mediated gene transfer were used to complement a hamster cytidine auxotroph deficient in CTP synthetase activity and eventually to clone human genomic and cDNA fragments coding for the structural gene. Our approach was to isolate human Alu+ fragments from a tertiary transfectant and to utilize these fragments to screen a panel of primary transfectants. In this manner two DNA fragments, both mapping within the structural gene, were identified and used to clone a partial length cDNA. The remaining portion of the open reading frame was obtained through the RACE polymerase chain reaction technique. The open reading frame encodes 591 amino acids having a striking degree of similarity to the Escherichia coli structural gene (48% identical amino acids with 76% overall similarity including conservative substitutions) with the glutamine amide transfer domain being particularly conserved. As regulatory mutations of CTP synthetase confer both multi-drug resistance to agents widely used in cancer chemotherapy and a mutator phenotype, the cloning of the structural gene will be important in assessing the relevance of such phenotypes to the development of cellular drug resistance. PMID:2113467

  18. 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

  19. A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol.

    PubMed

    Sajish, Mathew; Schimmel, Paul

    2015-03-19

    Resveratrol is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects by initiating a stress response that induces survival genes. Because human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) translocates to the nucleus under stress conditions, we considered the possibility that the tyrosine-like phenolic ring of resveratrol might fit into the active site pocket to effect a nuclear role. Here we present a 2.1 Å co-crystal structure of resveratrol bound to the active site of TyrRS. Resveratrol nullifies the catalytic activity and redirects TyrRS to a nuclear function, stimulating NAD(+)-dependent auto-poly-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1). Downstream activation of key stress signalling pathways are causally connected to TyrRS-PARP1-NAD(+) collaboration. This collaboration is also demonstrated in the mouse, and is specifically blocked in vivo by a resveratrol-displacing tyrosyl adenylate analogue. In contrast to functionally diverse tRNA synthetase catalytic nulls created by alternative splicing events that ablate active sites, here a non-spliced TyrRS catalytic null reveals a new PARP1- and NAD(+)-dependent dimension to the physiological mechanism of resveratrol. PMID:25533949

  20. Mechanism of oxidant-induced mistranslation by threonyl-tRNA synthetase.

    PubMed

    Wu, Jiang; Fan, Yongqiang; Ling, Jiqiang

    2014-06-01

    Aminoacyl-tRNA synthetases maintain the fidelity during protein synthesis by selective activation of cognate amino acids at the aminoacylation site and hydrolysis of misformed aminoacyl-tRNAs at the editing site. Threonyl-tRNA synthetase (ThrRS) misactivates serine and utilizes an editing site cysteine (C182 in Escherichia coli) to hydrolyze Ser-tRNA(Thr). Hydrogen peroxide oxidizes C182, leading to Ser-tRNA(Thr) production and mistranslation of threonine codons as serine. The mechanism of C182 oxidation remains unclear. Here we used a chemical probe to demonstrate that C182 was oxidized to sulfenic acid by air, hydrogen peroxide and hypochlorite. Aminoacylation experiments in vitro showed that air oxidation increased the Ser-tRNA(Thr) level in the presence of elongation factor Tu. C182 forms a putative metal binding site with three conserved histidine residues (H73, H77 and H186). We showed that H73 and H186, but not H77, were critical for activating C182 for oxidation. Addition of zinc or nickel ions inhibited C182 oxidation by hydrogen peroxide. These results led us to propose a model for C182 oxidation, which could serve as a paradigm for the poorly understood activation mechanisms of protein cysteine residues. Our work also suggests that bacteria may use ThrRS editing to sense the oxidant levels in the environment. PMID:24744241

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

    SciTech Connect

    Cichowicz, D.J.; Shane, B.

    1987-01-27

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

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

    PubMed

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

    2016-01-14

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

  3. Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae.

    PubMed Central

    Ausubel, F M; Bird, S C; Durbin, K J; Janssen, K A; Margolskee, R F; Peskin, A P

    1979-01-01

    Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase. PMID:40960

  4. 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

  5. Crystal Structure and Function of 5-Formaminoimidazole-4-carboxamide Ribonucleotide Synthetase from Methanocaldococcus jannaschii

    SciTech Connect

    Zhang, Yang; White, Robert H.; Ealick, Steven E.

    2008-08-06

    Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. PMID:27225077

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

    PubMed

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

    2013-01-18

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

  9. Search for primitive Methanopyrus based on genetic distance between Val- and Ile-tRNA synthetases.

    PubMed

    Yu, Zhiliang; Takai, Ken; Slesarev, Alexei; Xue, Hong; Wong, J Tze-Fei

    2009-10-01

    Since evidence indicates that the Last Universal Common Ancestor (LUCA) was phylogenetically closest to Methanopyrus kandleri among living organisms with elucidated genomes, this study has been directed to a search for the most primitive Methanopyrus lineage. For this purpose, the divergence of valyl-tRNA synthetase (ValRS) and isoleucyl-tRNA synthetase (IleRS) was employed as a measure of primitivity. Comparison of Methanopyrus kandleri and the Methanopyrus isolates GC34 and GC37 from the Pacific Ocean and KOL6, TAG1, TAG11, and SNP6 from the Atlantic Ocean established that the Pacific lineages are more primitive than the Atlantic lineages. Both the groups, however, are younger than environmental genomes from the Kairei Field of Central Indian Ridge in the Indian Ocean. These results showed that different Methanopyrus isolates differ significantly with respect to ValRS-IleRS divergence. On this basis, genomes giving rise to the ValRS and IleRS gene fragments from the Central Indian Ridge represent the most primitive Methanopyrus, phylogenetically the oldest living lineage closest to LUCA. PMID:19841848

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

    PubMed

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

    2016-06-14

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

  11. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs.

    PubMed

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-04-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes. PMID:24569352

  12. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

    PubMed Central

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J.; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-01-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes. PMID:24569352

  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. PMID:26464444

  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. 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

  16. MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

    PubMed Central

    Eriani, Gilbert; Karam, Joseph; Jacinto, Jomel; Morris Richard, Erin; Geslain, Renaud

    2015-01-01

    Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications. PMID:26067673

  17. Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?1

    PubMed Central

    Harrison, Judith; Pou de Crescenzo, Marie-Anne; Sené, Olivier; Hirel, Bertrand

    2003-01-01

    A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T1 progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated. PMID:12970491

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

    PubMed Central

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

    2016-01-01

    Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A>C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions. PMID:25227173

  19. Dominant lethality by expression of a catalytically inactive class I tRNA synthetase.

    PubMed Central

    Schmidt, E; Schimmel, P

    1993-01-01

    Alignment-guided mutagenesis was used to create an inactive, but toxic, aminoacyl-tRNA synthetase. An Asp-96-->Ala (D96A) replacement in the nucleotide binding fold of the class I Escherichia coli isoleucyl-tRNA synthetase inactivates the enzyme without disrupting its competence for binding isoleucine tRNA. Expression of plasmid-encoded mutant enzyme in a cell with a wild-type ileS chromosomal allele resulted in cell death. Introduction of a second K732T substitution previously shown to weaken tRNA binding gives an inactive D96A/K732T double mutant. Expression of the double mutant is not lethal to E. coli. D96A but not the double mutant significantly inhibited in vitro charging of isoleucine tRNA by the wild-type enzyme. The results suggest a dominant tRNA binding-dependent arrest of cell growth caused by a reduction in the pool of a specific tRNA. Specific tRNA binding drugs may have therapeutic applications for treatment of microbial pathogens. Images Fig. 1 Fig. 3 PMID:8346197

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

    PubMed Central

    Frawley, Elaine R.; Kranz, Robert G.

    2009-01-01

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

  1. Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus

    PubMed Central

    Shiyan, Anna; Thompson, Melanie; Köcher, Saskia; Tausendschön, Michaela; Santos, Helena; Hänelt, Inga; Müller, Volker

    2014-01-01

    Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated. PMID:24782854

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

    PubMed

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

    2014-12-23

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

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  4. Monoclonal antibodies against tyrosyl-tRNA synthetase and its isolated cytokine-like domain.

    PubMed

    Kondratiuk, Iuliia; Khoruzenko, Antonina; Cherednyk, Olga; Filonenko, Valeriy; Kornelyuk, Aleksander

    2013-06-01

    Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization. PMID:23750478

  5. A human leucyl-tRNA synthetase as an anticancer target

    PubMed Central

    Gao, Guangwei; Yao, Ying; Li, Kun; Mashausi, Dhahiri Saidi; Li, Dongsheng; Negi, Hema; Kamle, Suchitra; Chen, Hao; Wu, Zhenghua; Zhou, Huchen; Li, Dawei

    2015-01-01

    Several aminoacyl-tRNA synthetases have been reported to be overexpressed for charging essential aminoacyl-tRNAs in many cancer types. In this study, we aimed to explore the potential role of leucyl-tRNA synthetase (LARS) as an anticancer target. MTT assay was performed to screen inhibitors to human LARS (hsLARS) from compounds AN2690 and its derivatives, compounds 1–6, in U2OS and SKOV3 cells. The compound with the strongest inhibitory ability was further investigated for its inhibitory effect in cancer cell lines and in an animal tumor model. Additionally, a LARS-rescue experiment was performed to explore the potential target in U2OS using Western blot and flow cytometry. Luciferase reporter assay was designed to analyze the effect of of hsLARS inhibitor on p21 activation. We identified an hsLARS inhibitor (compound 2) that suppressed the proliferation of U2OS and SKOV3 cells in vitro. A LARS-rescue experiment demonstrated that the proliferation inhibition was induced by targeting intracellular LARS. In addition, the hsLARS inhibition was shown to activate the p21 early transcription and promote cell apoptosis, as well as reduce implanted EMT6 tumor progression in mice. Our results suggest that LARS might serve as a potential anticancer target through the p21 signaling pathway and that the nutritional signaling pathway may provide a valuable anticancer strategy for further investigation. PMID:26508878

  6. Fatty tumours of the uterus.

    PubMed Central

    Pounder, D J

    1982-01-01

    Uterine fatty tumours (UFT) are uncommon and have received little attention in the English literature. They have aroused interest as a consequence of occasional diagnostic confusion with sarcomas and the continuing unresolved dispute as to their histogenesis. Three cases of UFT are described and the pathological features of note discussed. The viewpoint that these tumours are hamartomas/choristomas is rejected. UFT most probably represent tumour metaplasia within a leiomyoma. There is no uniform accepted nomenclature for such tumours and it is suggested that they be designated "uterine fatty tumours" and subdivided into "lipoma" and "mixed lipoma/leiomyoma" (synonym lipoleiomyoma). Images PMID:7174848

  7. Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38.

    PubMed

    Rémion, Azaria; Khoder-Agha, Fawzi; Cornu, David; Argentini, Manuela; Redeker, Virginie; Mirande, Marc

    2016-07-01

    Human cytoplasmic lysyl-tRNA synthetase (LysRS) is associated within a multi-aminoacyl-tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work. PMID:27398309

  8. 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. PMID:25907574

  9. Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola.

    PubMed

    Li, Jiashan; Xia, Chengqiang; Fang, Xiaoran; Xue, Haizhao; Song, Xin

    2016-08-01

    The sophorolipid-producing strain Starmerella bombicola CGMCC 1576 has a remarkable ability to produce sophorolipids (SLs) under the acidic and lactonic forms with almost equal proportion. In this study, we found the gene encoding for the long-chain acyl-CoA synthetase (ALCS). This enzyme was putatively identified as a membrane-bound long-chain fatty acid transport protein and contributed to the uptake of long-chain fatty acids. Disruption of the alcs gene resulted in an impaired growth of the alcs-deleted mutant in minimal media containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the fluorescent-tagged long-chain fatty acid analogue-boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823). The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of the SLs produced by the gene-deleted mutant of ∆alcs::six showed almost no lactonic form of SLs, and the acidic SLs were composed of medium-chain. The ALCS enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with pMAL-c2x-alcs. The enzyme was purified through a maltose-binding protein (MBP) affinity chromatography column and was confirmed to be homogeneous by SDS-PAGE. The recombinant enzyme could catalyze the formation of the long-chain acyl-CoA when the long-chain fatty acids and the coenzyme A were used as substrates. PMID:27183996

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

    PubMed

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

    2014-01-01

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

  11. Expression of fatty acid synthase in nonalcoholic fatty liver disease

    PubMed Central

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD. PMID:20606731

  12. Expression of fatty acid synthase in nonalcoholic fatty liver disease.

    PubMed

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD. PMID:20606731

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

    SciTech Connect

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G.; Kuusksalu, A.; Kelve, M.

    1997-07-01

    Recently the authors established the presence of relatively high amounts of 2{prime}-5{prime} oligoadenylates (2{prime}-5{prime} A) and 2{prime}-5{prime} oligoadenylate synthetase (2{prime}-5{prime} A synthetase) in the marine sponge Geodia cydonium. Here they determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2{prime}-5{prime} A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2{prime}-5{prime} A content and the activity of 2{prime}-5{prime} A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26 C), cold shock (6 C), pH shock (pH 6), and hypertonic shock and subsequent incubation for 18 h under ambient conditions (16 C). No response was observed after exposure of sponges to an alkaline (pH 10) or hypotonic environment. Similar changes have been found for the expression of heat shock protein HSP70 in G. cydonium. These results show that 2{prime}-5{prime} A in sponges may be useful as a novel biomarker for environmental monitoring.

  14. Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

    SciTech Connect

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

    2010-03-01

    E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization.

  15. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain...

  16. ANALYSIS OF RILERS, AN ISOLEUCYL-TRNA SYNTHETASE GENE ASSOCIATED WITH MUPIROCIN PRODUCTION BY PSUEDOMONAS FLUORESCENS NCIMB 10586

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some strains of Pseudomonas fluorescens produce the antibiotic mupirocin, which functions as a competitive inhibitor of isoleucyl-tRNA synthetase (ILERS). Mupirocin-producing strains of P. fluorescens must overcome the inhibitory effects of the antibiotic to avoid self-suicide. However, it is not c...

  17. Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

    PubMed Central

    Borel, F; Vincent, C; Leberman, R; Härtlein, M

    1994-01-01

    Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs. Images PMID:8065908

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

    PubMed Central

    Bowman, Thomas A.; O'Keeffe, Kayleigh R.; D'Aquila, Theresa; Yan, Qing Wu; Griffin, John D.; Killion, Elizabeth A.; Salter, Deanna M.; Mashek, Douglas G.; Buhman, Kimberly K.; Greenberg, Andrew S.

    2016-01-01

    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 ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. Methods To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5−/−). Results Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5−/−, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5−/− had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5−/− mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (∼13-fold) were increased in ACSL5−/− as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5−/− mice as compared to control mice. Conclusions In summary, ACSL5−/− mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies

  19. Non-Alcoholic Fatty Liver Disease (NAFLD)

    MedlinePlus

    Non-Alcoholic Fatty Liver Disease What is Non-alcoholic fatty liver disease (NAFLD)? FAT N AFLD is a name that is given to a ... and under “Liver Health Information view ‘Nonalcoholic fatty liver Disease (NAFLD/NASH)’ IMPORTANT REMINDER: This information from the ...

  20. Dietary fatty acids and minerals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accumulating evidence in animals and humans shows that dietary fatty acids influence the absorption and utilization of certain mineral elements. Fat intake exceeding 10% of energy intake reduces calcium uptake and use by the body, and this effect is more pronounced with saturated compared to unsatu...

  1. Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels

    PubMed Central

    Muhitch, Michael J.

    1989-01-01

    Maize (Zea mays L.) kernel pedicels, including vascular tissues, pedicel parenchyma, placento-chalazal tissue, and the surrounding pericarp, contained two forms of glutamine synthetase (EC 6.3.1.2), separable by anion exchange chromatography under mildly acidic conditions. The earlier-eluting activity (GSp1), but not the later-eluting activity (GSp2), was chromatographically distinct from the maize leaf and root glutamine synthetases. The level of GSp1 activity changed in a developmentally dependent manner while GSp2 activity was constitutive. GSp1 and GSp2 exhibited distinct ratios of transferase to hydroxylamine-dependent synthetase activities (5 and 23, respectively), which did not change with kernel age. Purified pedicel glutamine synthetases had native relative molecular masses of 340,000, while the subunit relative molecular masses differed slightly at 38,900 and 40,500 for GSp1 and GSp2, respectively. Both GS forms required free Mg2+ with apparent Kms = 2.0 and 0.19 millimolar for GSp1 and GSp2, respectively. GSp1 had an apparent Km for glutamate of 35 millimolar and exhibited substrate inhibition at glutamate concentrations greater than 90 millimolar. In contrast, GSp2 exhibited simple Michaelis-Menten kinetics for glutamate with a Km value of 3.4 millimolar. Both isozymes exhibited positive cooperativity for ammonia, with S0.5 values of 100 and 45 micromolar, respectively. GSp1 appears to be a unique, kernel-specific form of plant glutamine synthetase. Possible functions for the pedicel GS isozymes in kernel nitrogen metabolism are discussed. Images Figure 4 PMID:16667150

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

    PubMed Central

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

    2003-01-01

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

  3. Nitrated fatty acids: Synthesis and measurement

    PubMed Central

    Woodcock, Steven R.; Bonacci, Gustavo; Gelhaus, Stacy L.; Schopfer, Francisco J.

    2012-01-01

    Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis, sample extraction from complex biological matrices, and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by LC-MS. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed. PMID:23200809

  4. Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

    PubMed

    Tamano, Koichi; Miura, Ai

    2016-09-01

    Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression. PMID:26998626

  5. Partial Purification and Properties of d-Desthiobiotin Synthetase from Escherichia coli1

    PubMed Central

    Cheeseman, P.; Pai, C. H.

    1970-01-01

    d-Desthiobiotin synthetase, an enzyme that catalyzes the synthesis of d-desthiobiotin from dl-7,8-diaminopelargonic acid and HCO3−, was purified 100-fold from cells of a biotin mutant strain of Escherichia coli. Adenosine triphosphate and Mg2+ were shown, especially in purified extracts, to be obligatory for enzyme activity, although concentrations higher than 5 mm caused severe inhibition of the reaction with unpurified cell-free extracts. Adenosine diphosphate and adenosine monophosphate were shown to inhibit the reaction, but fluoride (up to 50 mm) had no detectable effect. The product of the enzyme reaction was identical to d-desthiobiotin on the basis of biological activity and chromatography. Furthermore, when H14CO3− was used as a substrate, the radioactive product was shown to be 14C-desthiobiotin labeled exclusively in the ureido carbon. PMID:4923070

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

    PubMed

    Nausch, Henrik; Huckauf, Jana; Broer, Inge

    2016-02-01

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

  7. SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle.

    PubMed

    Nakagawa, Takashi; Lomb, David J; Haigis, Marcia C; Guarente, Leonard

    2009-05-01

    Sirtuins are NAD-dependent protein deacetylases that connect metabolism and aging. In mammals, there are seven sirtuins (SIRT1-7), three of which are associated with mitochondria. Here, we show that SIRT5 localizes in the mitochondrial matrix and interacts with carbamoyl phosphate synthetase 1 (CPS1), an enzyme, catalyzing the initial step of the urea cycle for ammonia detoxification and disposal. SIRT5 deacetylates CPS1 and upregulates its activity. During fasting, NAD in liver mitochondria increases, thereby triggering SIRT5 deacetylation of CPS1 and adaptation to the increase in amino acid catabolism. Indeed, SIRT5 KO mice fail to upregulate CPS1 activity and show elevated blood ammonia during fasting. Similar effects occur during long-term calorie restriction or a high protein diet. These findings demonstrate SIRT5 plays a pivotal role in ammonia detoxification and disposal by activating CPS1. PMID:19410549

  8. Detection of carbamyl phosphate synthetase 1 deficiency using duodenal biopsy samples.

    PubMed Central

    Hoogenraad, N J; Mitchell, J D; Don, N A; Sutherland, T M; Mc Leay, A C

    1980-01-01

    The activity of urea cycle enzymes was assayed in duodenal biopsy specimens obtained from a female infant who presented with neonatal hyperammonaemia. All enzyme levels were normal except N-acetyl glutamate-dependent carbamyl phosphate synthetase 1 (CPS1) which was half the mean activity in normal control specimens. A similar deficiency of CPS1 was also shown in duodenal specimens from the patient's mother who became slightly symptomatic after relatively high protein meals and during pregnancy, and had spontaneously modified her diet to one with protein restriction. The patient is growing normally on a dietary regimen similar to that spontaneously adopted by her mother. Urea cycle enzyme activity in the duodenal biopsy material from the controls was similar to that found in the normal human liver and appears to have distinct advantages as a means of assaying for urea cycle defects in patients with hyperammonaemia and their relatives. PMID:7416778

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

    SciTech Connect

    Langston-Unkefer, P.J.

    1991-06-11

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

  10. GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Ubiyvovk, Vira M; Nazarko, Taras Y; Stasyk, Olena G; Sohn, Min Jeong; Kang, Hyun Ah; Sibirny, Andrei A

    2002-08-01

    The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121. PMID:12702282

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

    PubMed Central

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

    2012-01-01

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

  12. 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.

  13. Thromboxane synthetase inhibition reduces ventricular irritability after coronary occlusion and reperfusion.

    PubMed

    Austin, J C; Berrizbeitia, L D; Schoen, F J; Kauffman, R P; Hechtman, H B; Cohn, L H

    1988-03-01

    Reperfusion of ischemic tissue is responsible for production of metabolites with deleterious local vascular effects. Thromboxane A2, a potent vasoconstrictor and platelet aggregator, has been implicated as a mediator of the "reperfusion injury." We studied the effect of an experimental thromboxane synthetase inhibitor, OKY-046, on coronary sinus thromboxane levels, ventricular irritability, myocardial contractility, infarct salvage, and histologic features of reperfusion. Sixteen sheep were randomized to OKY-046, 3 mg/kg, or saline vehicle before 3-hour occlusion and subsequent reperfusion of the left anterior descending artery. The OKY group demonstrated less ventricular irritability as measured by incidence of ventricular fibrillation and necessity for countershock to reverse tachyarrhythmias. Coronary sinus thromboxane levels were significantly lower in the OKY group compared with the control group. There is additional evidence to suggest that OKY increases infarct salvage and attenuates histologic features of microcirculatory damage. PMID:3344655

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

    NASA Astrophysics Data System (ADS)

    Rosson, Dan; Lewis, Charles A.; Ellis, Paul D.; Dunlap, R. Bruce

    1994-03-01

    Fluorotyrosine has been incorporated into thymidylate synthetase from Lactobacillus casei by growth of the bacterium in media containing 3-fluorotyrosine. The enzyme exhibited a specific activity 70% of that of the normal enzyme and formed a covalent binary complex with pyrimidine nucleotides, as well as a covalent ternary complex with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate. 19F nuclear magnetic resonance spectroscopy has been used to follow the formation of these complexes. 5-Fluorodeoxyuridylate, dUMP, dTMP and dCMP produced identical conformational changes in the enzyme as monitored by the fluorotyrosyl resonances. Ternary complex formation of the fluorotyrosine-containing enzyme with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate resulted in further spectral changes.

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

    PubMed

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

    2012-06-01

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

  16. Design, Synthesis and Biological Evaluation of Trypanosoma brucei Trypanothione Synthetase Inhibitors

    PubMed Central

    Spinks, Daniel; Torrie, Leah S; Thompson, Stephen; Harrison, Justin R; Frearson, Julie A; Read, Kevin D; Fairlamb, Alan H; Wyatt, Paul G; Gilbert, Ian H

    2012-01-01

    Trypanothione synthetase (TryS) is essential for the survival of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis. It is one of only a handful of chemically validated targets for T. brucei in vivo. To identify novel inhibitors of TbTryS we screened our in-house diverse compound library that contains 62 000 compounds. This resulted in the identification of six novel hit series of TbTryS inhibitors. Herein we describe the SAR exploration of these hit series, which gave rise to one common series with potency against the enzyme target. Cellular studies on these inhibitors confirmed on-target activity, and the compounds have proven to be very useful tools for further study of the trypanothione pathway in kinetoplastids. PMID:22162199

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

    PubMed Central

    Steinchen, Wieland; Lackner, Gerald; Yasmin, Sabiha; Schrettl, Markus; Dahse, Hans-Martin

    2013-01-01

    The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[32P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus. PMID:23974138

  18. A Nonribosomal Peptide Synthetase-derived Iron(III) Complex from the Pathogenic Fungus Aspergillus fumigatus

    PubMed Central

    Yin, Wen-Bing; Baccile, Joshua A.; Bok, Jin Woo; Chen, Yiming; Keller, Nancy P.; Schroeder, Frank C.

    2013-01-01

    Small molecules (SMs) play central roles as virulence factors of pathogenic fungi and bacteria; however, genomic analyses suggest that the majority of microbial SMs have remained uncharacterized. Based on microarray analysis followed by comparative metabolomics of overexpression/knockout mutants we identified a tryptophan-derived iron(III)-complex, hexadehydroastechrome (HAS), as the major product of the cryptic has non-ribosomal peptide synthetase (NRPS) gene cluster in the human pathogen Aspergillus fumigatus. Activation of the has cluster created a highly virulent A. fumigatus strain that increased mortality of infected mice. Comparative metabolomics of different mutant strains allowed to propose a pathway for HAS biosynthesis and further revealed cross-talk with another NRPS pathway producing the anti-cancer fumitremorgins. PMID:23360537

  19. Optimization of Inhibitors of Mycobacterium tuberculosis Pantothenate Synthetase Based on Group Efficiency Analysis

    PubMed Central

    Hung, Alvin W.; Silvestre, H. Leonardo; Wen, Shijun; George, Guillaume P. C.; Boland, Jennifer; Blundell, Tom L.; Ciulli, Alessio

    2015-01-01

    Abstract Ligand efficiency has proven to be a valuable concept for optimization of leads in the early stages of drug design. Taking this one step further, group efficiency (GE) evaluates the binding efficiency of each appendage of a molecule, further fine‐tuning the drug design process. Here, GE analysis is used to systematically improve the potency of inhibitors of Mycobacterium tuberculosis pantothenate synthetase, an important target in tuberculosis therapy. Binding efficiencies were found to be distributed unevenly within a lead molecule derived using a fragment‐based approach. Substitution of the less efficient parts of the molecule allowed systematic development of more potent compounds. This method of dissecting and analyzing different groups within a molecule offers a rational and general way of carrying out lead optimization, with potential broad application within drug discovery. PMID:26486566

  20. 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.

  1. 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

  2. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation. PMID:26990986

  3. Structural basis of improved second-generation 3-nitro-tyrosine tRNA synthetases.

    PubMed

    Cooley, Richard B; Feldman, Jessica L; Driggers, Camden M; Bundy, Taylor A; Stokes, Audrey L; Karplus, P Andrew; Mehl, Ryan A

    2014-04-01

    Genetic code expansion has provided the ability to site-specifically incorporate a multitude of noncanonical amino acids (ncAAs) into proteins for a wide variety of applications, but low ncAA incorporation efficiency can hamper the utility of this powerful technology. When investigating proteins containing the post-translational modification 3-nitro-tyrosine (nitroTyr), we developed second-generation amino-acyl tRNA synthetases (RS) that incorporate nitroTyr at efficiencies roughly an order of magnitude greater than those previously reported and that advanced our ability to elucidate the role of elevated cellular nitroTyr levels in human disease (e.g., Franco, M. et al. Proc. Natl. Acad. Sci. U.S.A 2013 , 110 , E1102 ). Here, we explore the origins of the improvement achieved in these second-generation RSs. Crystal structures of the most efficient of these synthetases reveal the molecular basis for the enhanced efficiencies observed in the second-generation nitroTyr-RSs. Although Tyr is not detectably incorporated into proteins when expression media is supplemented with 1 mM nitroTyr, a major difference between the first- and second-generation RSs is that the second-generation RSs have an active site more compatible with Tyr binding. This feature of the second-generation nitroTyr-RSs appears to be the result of using less stringent criteria when selecting from a library of mutants. The observation that a different selection strategy performed on the same library of mutants produced nitroTyr-RSs with dramatically improved efficiencies suggests the optimization of established selection protocols could lead to notable improvements in ncAA-RS efficiencies and thus the overall utility of this technology. PMID:24611875

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

    PubMed

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

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  5. Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

    PubMed

    van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

    2015-06-01

    Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. PMID:25736594

  6. A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading

    PubMed Central

    Zhou, Xiao-Long; Ruan, Zhi-Rong; Wang, Meng; Fang, Zhi-Peng; Wang, Yong; Chen, Yun; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2014-01-01

    Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNAThr2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNAThr1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNAThrs during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNAThr2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNAThr1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNAThr1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core. PMID:25414329

  7. A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading.

    PubMed

    Zhou, Xiao-Long; Ruan, Zhi-Rong; Wang, Meng; Fang, Zhi-Peng; Wang, Yong; Chen, Yun; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2014-12-16

    Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNA(Thr)2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNA(Thr)1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNA(Thr)s during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNA(Thr)2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNA(Thr)1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNA(Thr)1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core. PMID:25414329

  8. On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli.

    PubMed

    Yee, M C; Horn, V; Yanofsky, C

    1996-06-21

    The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0. PMID:8662916

  9. Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase.

    PubMed

    Burke, B; Lipman, R S; Shiba, K; Musier-Forsyth, K; Hou, Y M

    2001-06-01

    Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine. PMID:11342535

  10. Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

    PubMed

    Schulze, Jörg O; Masoumi, Ava; Nickel, Daniel; Jahn, Martina; Jahn, Dieter; Schubert, Wolf-Dieter; Heinz, Dirk W

    2006-09-01

    Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity. PMID:16876193

  11. Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase.

    PubMed

    Sherman, J M; Thomann, H U; Söll, D

    1996-03-15

    The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-specific protein-tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNAGln. Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNAGln. Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non- cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain ot make more productive interactions. PMID:8601833

  12. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

    PubMed Central

    Lam, H M; Peng, S S; Coruzzi, G M

    1994-01-01

    Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen. PMID:7846154

  13. Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing*

    PubMed Central

    Cvetesic, Nevena; Dulic, Morana; Bilus, Mirna; Sostaric, Nikolina; Lenhard, Boris; Gruic-Sovulj, Ita

    2016-01-01

    Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 103-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability. PMID:26921320

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

    PubMed Central

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

    2011-01-01

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

  15. Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase.

    PubMed

    Nakanishi, Kotaro; Bonnefond, Luc; Kimura, Satoshi; Suzuki, Tsutomu; Ishitani, Ryuichiro; Nureki, Osamu

    2009-10-22

    Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5' leader and 3' trailer sequences, removal of introns and addition of the CCA terminus. Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNA(Ile2) lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNA(Ile2) (refs 4-9). This modification switches tRNA(Ile2) from a methionine-specific to an isoleucine-specific tRNA. However, the aminoacylation of tRNA(Ile2) by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNA(Ile2) in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNA(Ile2) isolated from RNase-E-deficient Escherichia coli and did not detect mature tRNA(Ile2) lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNA(Ile2) through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNA(Ile2) by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis. PMID:19847269

  16. Purification, Characterization, and Expression of Multiple Glutamine Synthetases from Prevotella ruminicola 23

    PubMed Central

    Kim, Jong Nam; Cann, Isaac K. O.

    2012-01-01

    The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa. Optimal conditions for γ-glutamyl transferase activity were found to be 35°C at pH 5.6 with 0.25 mM Mn2+ ions (GSI) or 37°C at pH 6.0 (GSIII-1 and GSIII-2) with 0.50 to 1.00 mM Mn2+ ions. GSIII biosynthetic activity was found to be optimal at 50 to 60°C and pH 6.8 to 7.0 with 10 mM Mn2+ ions, while GSI displayed no GS biosynthetic activity. Kinetic analysis revealed Km values for glutamate and ammonium as well as for hydrolysis of ATP to be 8.58, 0.48, and 1.91 mM, respectively, for GSIII-1 and 1.72, 0.43, and 2.65 mM, respectively, for GSIII-2. A quantitative reverse transcriptase PCR assay (qRT-PCR) revealed GSIII-2 to be significantly induced by high concentrations of ammonia, and this corresponded with increases in measured GS activity. Collectively, these results show that both GSIII enzymes in P. ruminicola 23 are functional and indicate that GSIII-2, flanked by GOGAT (gltB and gltD genes), plays an important role in the acquisition and metabolism of ammonia, particularly under nonlimiting ammonia growth conditions. PMID:22020637

  17. New radiohalogenated alkenyl tellurium fatty acids

    SciTech Connect

    Srivastava, P.C.; Knapp, F.F. Jr.; Kabalka, G.W.

    1987-01-01

    Radiolabeled long-chain fatty acids have diagnostic value as radiopharmaceutical tools in myocardial imaging. Some applications of these fatty acids are limited due to their natural metabolic degradation in vivo with subsequent washout of the radioactivity from the myocardium. The identification of structural features that will increase the myocardial residence time without decreasing the heart uptake of long-chain fatty acids is of interest. Fatty acids containing the tellurium heteroatom were the first modified fatty acids developed that show unique prolonged myocardial retention and low blood levels. Our detailed studies with radioiodinated vinyliodide substituted tellurium fatty acids demonstrate that heart uptake is a function of the tellurium position. New techniques of tellurium and organoborane chemistry have been developed for the synthesis of a variety of radioiodinated iodoalkenyl tellurium fatty acids. 9 refs., 3 figs., 2 tabs.

  18. Fatty acid uptake in normal human myocardium

    SciTech Connect

    Vyska, K.; Meyer, W.; Stremmel, W.; Notohamiprodjo, G.; Minami, K.; Machulla, H.J.; Gleichmann, U.; Meyer, H.; Koerfer, R. )

    1991-09-01

    Fatty acid binding protein has been found in rat aortic endothelial cell membrane. It has been identified to be a 40-kDa protein that corresponds to a 40-kDa fatty acid binding protein with high affinity for a variety of long chain fatty acids isolated from rat heart myocytes. It is proposed that this endothelial membrane fatty acid binding protein might mediate the myocardial uptake of fatty acids. For evaluation of this hypothesis in vivo, influx kinetics of tracer-labeled fatty acids was examined in 15 normal subjects by scintigraphic techniques. Variation of the plasma fatty acid concentration and plasma perfusion rate has been achieved by modulation of nutrition state and exercise conditions. The clinical results suggest that the myocardial fatty acid influx rate is saturable by increasing fatty acid plasma concentration as well as by increasing plasma flow. For analysis of these data, functional relations describing fatty acid transport from plasma into myocardial tissue in the presence and absence of an unstirred layer were developed. The fitting of these relations to experimental data indicate that the free fatty acid influx into myocardial tissue reveals the criteria of a reaction on a capillary surface in the vicinity of flowing plasma but not of a reaction in extravascular space or in an unstirred layer and that the fatty acid influx into normal myocardium is a saturable process that is characterized by the quantity corresponding to the Michaelis-Menten constant, Km, and the maximal velocity, Vmax, 0.24 {plus minus} 0.024 mumol/g and 0.37 {plus minus} 0.013 mumol/g(g.min), respectively. These data are compatible with a nondiffusional uptake process mediated by the initial interaction of fatty acids with the 40-kDa membrane fatty acid binding protein of cardiac endothelial cells.

  19. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

    Imker, Heidi J.; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T.

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  20. Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols

    PubMed Central

    2013-01-01

    Background Recent progress in production of various biofuel precursors and molecules, such as fatty acids, alcohols and alka(e)nes, is a significant step forward for replacing the fossil fuels with renewable fuels. A two-step process, where fatty acids from sugars are produced in the first step and then converted to corresponding biofuel molecules in the second step, seems more viable and attractive at this stage. We have engineered an Escherichia coli strain to take care of the second step for converting short chain fatty acids into corresponding alcohols by using butyrate kinase (Buk), phosphotransbutyrylase (Ptb) and aldehyde/alcohol dehydrogenase (AdhE2) from Clostridium acetobutylicum. Results The engineered E. coli was able to convert butyric acid and other short chain fatty acids of chain length C3 to C7 into corresponding alcohols and the efficiency of conversion varied with different E. coli strain type. Glycerol proved to be a better donor of ATP and electron as compared to glucose for converting butyric acid to butanol. The engineered E. coli was able to tolerate up to 100 mM butyric acid and produced butanol with the conversion rate close to 100% under anaerobic condition. Deletion of native genes, such as fumarate reductase (frdA) and alcohol dehydrogenase (adhE), responsible for side products succinate and ethanol, which act as electron sink and could compete with butyric acid uptake, did not improve the butanol production efficiency. Indigenous acyl-CoA synthetase (fadD) was found to play no role in the conversion of butyric acid to butanol. Engineered E. coli was cultivated in a bioreactor under controlled condition where 60 mM butanol was produced within 24 h of cultivation. A continuous bioreactor with the provision of cell recycling allowed the continuous production of butanol at the average productivity of 7.6 mmol/l/h until 240 h. Conclusions E. coli engineered with the pathway from C. acetobutylicum could efficiently convert butyric acid

  1. Variable Sensitivity to Bacterial Methionyl-tRNA Synthetase Inhibitors Reveals Subpopulations of Streptococcus pneumoniae with Two Distinct Methionyl-tRNA Synthetase Genes

    PubMed Central

    Gentry, Daniel R.; Ingraham, Karen A.; Stanhope, Michael J.; Rittenhouse, Stephen; Jarvest, Richard L.; O'Hanlon, Peter J.; Brown, James R.; Holmes, David J.

    2003-01-01

    As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, ≥8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors. PMID:12760849

  2. 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

  3. Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

    PubMed Central

    Tarassov, I; Entelis, N; Martin, R P

    1995-01-01

    Cytoplasmic tRNA(Lys)CUU is the only nuclear-encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl-tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl-tRNA synthetase is able to form specific and stable RNP complexes with the amino-acylated tRNA. Furthermore, an N-terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s). Images PMID:7628447

  4. 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.

  5. 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

  6. Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by Microarray-Bioinformatics analysis.

    PubMed

    Yeh, Ching-Sheng; Wang, Jaw-Yuan; Cheng, Tian-Lu; Juan, Chin-Hung; Wu, Chan-Han; Lin, Shiu-Ru

    2006-02-28

    The present study systematically explored metabolic pathways and altered expressions of genes speculatively participating in colorectal carcinogenesis by using a Microarray-Bioinformatic analysis methods. The results revealed that 157 genes were up-regulated and 281 genes were down-regulated in colorectal cancer (CRC). Gene Ontology (GO) and relevant bioinformatics tools indicated that the functional category to which 438 genes (12%; 438/3800) of the most frequent alteration belonged was metabolism. The analysis of 10 colorectal cancer tissue specimens demonstrated that genes involved in fatty acid metabolic pathways had high rates of overexpression. In addition, we stimulated CRL-1790 cell line with linoleic acid (a polyunsaturated fatty acid) for 12, 24, 48 and 72 h. Cell proliferation was elevated by 5, 25, 28 and 31% (P<0.05), respectively. Further analyses revealed that the genes increasingly expressed in the cell line included enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase (EHHADH), enoyl Coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1); glutaryl-Coenzyme A dehydrogenase (GCDH), acyl-Coenzyme A oxidase 2, branched chain (ACOX2); acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor (ACADS); carnitine palmitoyltransferase 1B (CPT1B), acyl-CoA synthetase long-chain family member 5 (ACSL5), and cytochrome P450, family 4, subfamily A, and polypeptide 11 (CYP4A11) genes. This indicated that the stimulating effect of linoleic acid on cell proliferation was due to interference with the metabolic pathway of fatty acid metabolism. In conclusion, genes with altered expression levels in CRC were mainly associated with fatty acid metabolic pathways speculated to have an important role linked to carcinogenesis. PMID:15885896

  7. Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases.

    PubMed Central

    Eriani, G; Dirheimer, G; Gangloff, J

    1990-01-01

    By screening of an Escherichia coli plasmidic library using antibodies against aspartyl-tRNA synthetase (AspRS) several clones were obtained containing aspS, the gene coding for AspRS. We report here the nucleotide sequence of aspS and the corresponding primary structure of the aspartyl-tRNA synthetase, a protein of 590 amino acid residues with a Mr 65,913, a value in close agreement with that observed for the purified protein. Primer extension analysis of the aspS mRNA using reverse transcriptase located its 5'-end at 94 nucleotides upstream of the translation initiation AUG; nuclease S1 analysis located the 3'-end at 126 nucleotides downstream of the stop codon UGA. Comparison of the DNA-derived protein sequence with known aminoacyl-tRNA sequences revealed important homologies with asparaginyl- and lysyl-tRNA synthetases from E.coli; more than 25% of their amino acid residues are identical, the homologies being distributed preferencially in the first part and the carboxy-terminal end of the molecule. Mutagenesis directed towards a consensus tetrapeptide (Gly-Leu-Asp-Arg) and the carboxy-terminal end showed that both domains could be implicated in catalysis as well as in ATP binding. Images PMID:2129559

  8. 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

  9. 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. PMID:27197800

  10. Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans.

    PubMed

    Tiwary, B N; Bisen, P S; Sinha, U

    1987-08-01

    We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase. PMID:3312953

  11. Severe respiratory failure as a presenting feature of an interstitial lung disease associated with anti-synthetase syndrome (ASS).

    PubMed

    Piroddi, Ines Maria Grazia; Ferraioli, Gianluca; Barlascini, Cornelius; Castagneto, Corrado; Nicolini, Antonello

    2016-07-01

    Anti-synthetase syndrome (ASS) is defined as a heterogeneous connective tissue disorder characterized by the association of an interstitial lung disease (ILD) with or without inflammatory myositis with the presence of anti-aminoacyl-tRNA-synthetase antibodies. ILD is one of the major extra-muscular manifestations of polymyositis and dermatomyositis. We report a case of a patient with dyspnea, cough, and intermittent fever as well as ILD associated ASS in the absence of muscular involvement. This patient was admitted to the emergency department with severe respiratory failure requiring non-invasive ventilation. Our patient's case demonstrates that the diagnosis of ASS may not be obvious. However, its diagnosis leads to appropriate and potentially life-saving treatment. PMID:27424829

  12. Diagnostic of protein crystallization by dynamic light scattering; an application to an aminoacyl-tRNA synthetase

    NASA Astrophysics Data System (ADS)

    Mikol, Vincent; Vincendon, Pascale; Eriani, Gilbert; Hirsch, Ernest; Giegé, Richard

    1991-03-01

    The apparent hydrodynamic radius of a truncated form of baker's yeast aspartyl-tRNA synthetase has been measured in various precipitating agent solutions as a function of the protein concentration by dynamic light scattering. In solvents containing ammonium sulfate or 2-methyl-2,4-pentanediol as the precipitating agent the protein remains essentially monodisperse, whereas in the presence of polyethylene glycol interactions and aggregations between protein molecules are detected before reaching supersaturation. These data are indications of possible crystallizations of the protein by the two former precipitants and no crystallization by the latter one. Crystallization experiments indeed have shown that the truncated synthetase crystallizes in the presence of ammonium sulfate and that no crystals grow in solvents containing polyethylene glycol.

  13. Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

    PubMed Central

    Sauter, Claude; Lorber, Bernard; Gaudry, Agnès; Karim, Loukmane; Schwenzer, Hagen; Wien, Frank; Roblin, Pierre; Florentz, Catherine; Sissler, Marie

    2015-01-01

    Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation. PMID:26620921

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

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-01

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  15. Diminished acyl-CoA synthetase isoform 4 activity in INS 832/13 cells reduces cellular epoxyeicosatrienoic acid levels and results in impaired glucose-stimulated insulin secretion.

    PubMed

    Klett, Eric L; Chen, Shufen; Edin, Matthew L; Li, Lei O; Ilkayeva, Olga; Zeldin, Darryl C; Newgard, Christopher B; Coleman, Rosalind A

    2013-07-26

    Glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is potentiated by fatty acids (FA). The initial step in the metabolism of intracellular FA is the conversion to acyl-CoA by long chain acyl-CoA synthetases (Acsls). Because the predominantly expressed Acsl isoforms in INS 832/13 cells are Acsl4 and -5, we characterized the role of these Acsls in beta-cell function by using siRNA to knock down Acsl4 or Acsl5. Compared with control cells, an 80% suppression of Acsl4 decreased GSIS and FA-potentiated GSIS by 32 and 54%, respectively. Knockdown of Acsl5 did not alter GSIS. Acsl4 knockdown did not alter FA oxidation or long chain acyl-CoA levels. With Acsl4 knockdown, incubation with 17 mm glucose increased media epoxyeicosatrienoic acids (EETs) and reduced cell membrane levels of EETs. Further, exogenous EETs reduced GSIS in INS 832/13 cells, and in Acsl4 knockdown cells, an EET receptor antagonist partially rescued GSIS. These results strongly suggest that Acsl4 activates EETs to form EET-CoAs that are incorporated into glycerophospholipids, thereby sequestering EETs. Exposing INS 832/13 cells to arachidonate or linoleate reduced Acsl4 mRNA and protein expression and reduced GSIS. These data indicate that Acsl4 modulates GSIS by regulating the levels of unesterified EETs and that arachidonate controls the expression of its activator Acsl4. PMID:23766516

  16. 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

  17. Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus.

    PubMed

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

    2012-05-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

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

    PubMed Central

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

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

  19. Fatty acids of Pinus elliottii tissues.

    NASA Technical Reports Server (NTRS)

    Laseter, J. L.; Lawler, G. C.; Walkinshaw, C. H.; Weete, J. D.

    1973-01-01

    The total fatty constituents of slash pine (Pinus elliottii) tissue cultures, seeds, and seedlings were examined by GLC and MS. Qualitatively, the fatty acid composition of these tissues was found to be very similar to that reported for other pine species. The fatty acid contents of the tissue cultures resembled that of the seedling tissues. The branched-chain C(sub 17) acid reported for several other Pinus species was confirmed as the anteiso isomer.

  20. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sucrose fatty acid esters. 172.859 Section 172.859... CONSUMPTION Multipurpose Additives § 172.859 Sucrose fatty acid esters. Sucrose fatty acid esters identified...) Sucrose fatty acid esters are the mono-, di-, and tri-esters of sucrose with fatty acids and are...