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Sample records for human holocarboxylase synthetase

  1. 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

  2. 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 ...

  3. The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

    PubMed Central

    Hassan, Yousef I.; Moriyama, Hideaki; Zempleni, Janos

    2010-01-01

    Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed. PMID:20026029

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

    SciTech Connect

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

    2011-08-19

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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. DROSOPHILA HOLOCARBOXYLASE SYNTHETASE IS A CHROMOSOMAL PROTEIN REQUIRED FOR NORMAL HISTONE BIOTINYLATION, GENE TRANSCRIPTION PATTERNS, LIFESPAN AND HEAT TOLERANCE12

    PubMed Central

    Camporeale, Gabriela; Giordano, Ennio; Rendina, Rosaria; Zempleni, Janos; Eissenberg, Joel C.

    2006-01-01

    Posttranslational modifications of histones play important roles in chromatin structure and genomic stability. Distinct lysine residues in histones are targets for covalent binding of biotin, catalyzed by holocarboxylase synthetase (HCS) and biotinidase (BTD). Histone biotinylation has been implicated in heterochromatin structures, DNA repair, and mitotic chromosome condensation. To test whether HCS and BTD deficiency alters histone biotinylation, and to characterize phenotypes associated with HCS and BTD deficiency, HCS- and BTD-deficient flies were generated by RNA interference (RNAi). Expression of HCS and BTD decreased by 65–90% in RNAi-treated flies, as judged by mRNA abundance, BTD activity, and abundance of HCS protein. Decreased expression of HCS and BTD caused decreased biotinylation of K9 and K18 in histone H3. This was associated with altered expression of 201 genes in HCS-deficient flies. Lifespan of HCS- and BTD-deficient flies decreased by up to 32% compared to wild-type controls. Heat tolerance decreased by up to 55% in HCS-deficient flies compared to controls, as judged by survival times; effects of BTD deficiency were minor. Consistent with this observation, HCS deficiency was associated with altered expression of 285 heat-responsive genes. HCS and BTD deficiency did not affect cold tolerance, suggesting stress-specific effects of chromatin remodeling by histone biotinylation. This is the first study to provide evidence that HCS-dependent histone biotinylation affects gene function and phenotype, suggesting that the complex phenotypes of HCS- and BTD-deficiency disorders may reflect chromatin structure changes. PMID:17056793

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. Biotinyl-methyl 4-(amidomethyl) benzoate is a competitive inhibitor of human biotinidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Posttranslational modification of histones by biotinylation can be catalyzed by both biotinidase (BTD) and holocarboxylase synthetase (HCS). Biotinylation of histones is an important epigenetic mechanism to regulate gene expression, DNA repair, and chromatin remodeling. The role of BTD in histone ...

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis.

    PubMed

    de Cima, Sergio; Polo, Luis M; Díez-Fernández, Carmen; Martínez, Ana I; Cervera, Javier; Fita, Ignacio; Rubio, Vicente

    2015-01-01

    Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. PMID:26592762

  11. Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis

    PubMed Central

    de Cima, Sergio; Polo, Luis M.; Díez-Fernández, Carmen; Martínez, Ana I.; Cervera, Javier; Fita, Ignacio; Rubio, Vicente

    2015-01-01

    Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. PMID:26592762

  12. 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

  13. 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

  14. 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

  15. 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

  16. Expression of acyl-CoA synthetase 5 in human epidermis.

    PubMed

    Gaisa, N T; Köster, J; Reinartz, A; Ertmer, K; Ehling, J; Raupach, K; Perez-Bouza, A; Knüchel, R; Gassler, N

    2008-04-01

    The human epidermis is characterized by a constant renewal of keratinocytes embedded in a matrix enriched with lipids. Numerous proteins involved in lipid metabolism are found in human epidermis, especially in keratinocytes. Long-chain acyl-CoA derivatives, which are catalyzed by human ACSL5, are important metabolites in several biochemical pathways, including ceramide de novo synthesis. The aim of the present study was to investigate expression of acyl-CoA synthetase isoform 5 (ACSL5) in human epidermis by an in situ, as well as a molecular approach. We show that ACSL5 mRNA and protein are found in human epidermis, as well as in non-differentiated and differentiated HaCaT cells. Keratinocytes of stratum spinosum are the main source for ACSL5 expression in both meshed facial or abdominal skin and ridged skin of upper or lower extremities including TUNEL-positive cells in upper cellular layers. Single keratinocytes of chronic solar-exposed meshed facial epidermis occasionally display a stronger ACSL5 immunostaining. In conclusion, our study indicates that epidermal ACSL5 expression might be involved in differentiation and the stress response of keratinocytes. PMID:18228202

  17. A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

    PubMed

    Ikeuchi, Hideyuki; Meyer, Megan E; Ding, Yun; Hiratake, Jun; Richards, Nigel G J

    2009-09-15

    The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability. PMID:19683931

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

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

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

  19. Cyclic Peptide Inhibitors of HIV-1 Capsid-Human Lysyl-tRNA Synthetase Interaction

    PubMed Central

    2012-01-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNALys,3, which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents. PMID:22276994

  20. 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.

  1. 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

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

    SciTech Connect

    Kim, Jin Young; Lee, Kwang Hee; Shim, Myoung Sup; Shin, Hyein; Xu, Xue-Ming; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2010-06-18

    Selenophosphate synthetase 1 (SPS1) is an essential cellular gene in higher eukaryotes. Five alternative splice variants of human SPS1 (major type, {Delta}E2, {Delta}E8, +E9, +E9a) were identified wherein +E9 and +E9a make the same protein. The major type was localized in both the nuclear and plasma membranes, and the others in the cytoplasm. All variants form homodimers, and in addition, the major type forms a heterodimer with {Delta}E2, and {Delta}E8 with +E9. The level of expression of each splice variant was different in various cell lines. The expression of each alternative splice variant was regulated during the cell cycle. The levels of the major type and {Delta}E8 were gradually increased until G2/M phase and then gradually decreased. {Delta}E2 expression peaked at mid-S phase and then gradually decreased. However, +E9/+E9a expression decreased gradually after cell cycle arrest. The possible involvement of SPS1 splice variants in cell cycle regulation is discussed.

  3. Degenerate connective polypeptide 1 (CP1) domain from human mitochondrial leucyl-tRNA synthetase.

    PubMed

    Ye, Qing; Wang, Meng; Fang, Zhi-Peng; Ruan, Zhi-Rong; Ji, Quan-Quan; Zhou, Xiao-Long; Wang, En-Duo

    2015-10-01

    The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with noncognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain that has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different noncognate amino acids are not completely understood. Here, we identified the essential role of the evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, a broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for noncognate norvaline (Nva) and valine (Val). hmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Moreover, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity and acquired fidelity during aminoacylation. This work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency. PMID:26272616

  4. 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

  5. Methionine synthetase activity of human lymphocytes both replete in and depleted of vitamin B12.

    PubMed

    Hall, C A; Begley, J A; Chu, R C

    1986-10-01

    The activity of the enzyme methionine synthetase (MS) (methyltetrahydrofolate:homocysteine methyltransferase) (EC 2.1.1.13) was measured in human lymphocytes of various types and cobalamin (vitamin B12) status. Total and holo MS activity was low in unstimulated peripheral blood lymphocytes from persons with tissue deficiency of cobalamin, but not in cells from those with low serum cobalamin levels for other reasons. The MS activity of the lymphocyte was increased by treatment of the patients with vitamin B12. The number of lymphocytes was often low or low normal in the circulation of those deficient in cobalamin. Holo MS activity was low in an established line of human B cells, RPMI 6410 cells, depleted of cobalamin. The total and holo MS activity of both RPMI 6410 cells, replete or depleted, and lymphocytes stimulated in culture was increased by cobalamin in vitro; 222 nmol/L free cobalamin was roughly the equivalent of 0.22 nmol/L cobalamin bound to transcobalamin II. Both lymphocytes and RPMI 6410 cells required folate for growth and could meet these needs via methylfolate, homocysteine, and the cobalamin-dependent MS reaction. Depleted RPMI 6410 cells, however, used cobalamin in some way in addition to the provision of available folate from methylfolate. The consequences of the reduced MS activity in deficient cells could include a reduction in available folate with diminished capacity for clonal expansion of lymphocytes in reaction to infection and impairment of essential methylations including those of protein synthesis. The prompt induction of MS activity by cobalamin, especially in the in vitro model, suggests an effect of therapeutic vitamin B12 well in advance of the numerical increase in cells of the blood. PMID:3760673

  6. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

    SciTech Connect

    Guo, Min; Ignatov, Michael; Musier-Forsyth, Karin; Schimmel, Paul; Yang, Xiang-Lei

    2008-09-17

    In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The {alpha}{sub 2} homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that {alpha}{sub 2} LysRS is tightly bound into the core of the MSC with homodimeric {beta}{sub 2} p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive {alpha}{sub 2}{beta}{sub 2} heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-{angstrom} crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryote-specific sequences, one in the catalytic- and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein-protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.

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

    SciTech Connect

    Liu Honglin; Peng, Xiaohui; Zhao Fang; Zhang Guobin; Tao Ye; Luo Zhaofeng; Li Yang; Teng Maikun; Li Xu Wei Shiqiang

    2009-02-20

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of {alpha}-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO{sub 4}{sup 3-} allosteric site where PO{sub 4}{sup 3-} functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

  8. 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

  9. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    SciTech Connect

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  10. Conservation of structure in the human gene encoding argininosuccinate synthetase and the argG genes of the archaebacteria Methanosarcina barkeri MS and Methanococcus vannielii

    SciTech Connect

    Morris, C.J.; Reeve, J.N.

    1988-07-01

    The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomyces cerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.

  11. 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

  12. A Loss-of-Function Variant in the Human Histidyl-tRNA Synthetase (HARS) Gene is Neurotoxic In Vivo

    PubMed Central

    Vester, Aimee; Velez-Ruiz, Gisselle; McLaughlin, Heather M.; Lupski, James R.; Talbot, Kevin; Vance, Jeffery M.; Züchner, Stephan; Roda, Ricardo H.; Fischbeck, Kenneth H.; Biesecker, Leslie G.; Nicholson, Garth; Beg, Asim; Antonellis, Anthony

    2012-01-01

    Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed. PMID:22930593

  13. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  14. Identification of lethal mutations in yeast threonyl-tRNA synthetase revealing critical residues in its human homolog.

    PubMed

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-16

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  15. Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

    PubMed Central

    Alexandrova, Jana; Paulus, Caroline; Rudinger-Thirion, Joëlle; Jossinet, Fabrice; Frugier, Magali

    2015-01-01

    The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5′-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases. PMID:26327585

  16. Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

    PubMed Central

    Gowher, Ali; Smirnov, Alexandre; Tarassov, Ivan; Entelis, Nina

    2013-01-01

    In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNALysCUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNALysCUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms. PMID:23799079

  17. A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects.

    PubMed

    Wang, Yong; Zhou, Xiao-Long; Ruan, Zhi-Rong; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2016-03-18

    Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs. PMID:26811336

  18. A new γ-interferon-inducible promoter and splice variants of an anti-angiogenic human tRNA synthetase

    PubMed Central

    Liu, Jianming; Shue, Eveline; Ewalt, Karla L.; Schimmel, Paul

    2004-01-01

    Two forms of human tryptophanyl-tRNA synthetase (TrpRS) are produced in vivo through alternative mRNA splicing. The two forms, full-length TrpRS and mini TrpRS, are catalytically active, but are distinguished by the striking anti-proliferative and anti-angiogenic activity specific to mini TrpRS. Here we describe two new splice variants of human TrpRS mRNA. Their production was strongly regulated by γ-interferon (IFN-γ), an anti-proliferative cytokine known to stimulate the expression of other anti-angiogenic factors. A new IFN-γ-sensitive promoter was demonstrated to drive production of these splice variants. In human endothelial cells, both the newly discovered and a previously reported promoter were shown to respond specifically to IFN-γ and not to other cytokines such as tumor necrosis factor-α, transforming growth factor-β, interleukin-4 or erythropoietin. In addition, both promoters were stimulated by the ‘downstream’ interferon regulatory factor 1 that, in turn, is known to be regulated by the ‘upstream’ signal transducer and activator of transcription 1α subunit. Thus, the tandem promoters provide a dual system to regulate expression and alternative splicing of human TrpRS in vivo. PMID:14757836

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

    PubMed Central

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

    2011-01-01

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

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

    SciTech Connect

    Nichols, R.C.; Blinder, J.; Pai, S.I.

    1996-08-15

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

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

    PubMed

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

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma. PMID:25858017

  2. Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells

    PubMed Central

    Golej, Deidre L.; Askari, Bardia; Kramer, Farah; Barnhart, Shelley; Vivekanandan-Giri, Anuradha; Pennathur, Subramaniam; Bornfeldt, Karin E.

    2011-01-01

    Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E2 (PGE2) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE2. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE2 secretion. Thus, ACSL4 modulates PGE2 release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall. PMID:21242590

  3. 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

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

    SciTech Connect

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

    2009-06-04

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

  5. Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome

    PubMed Central

    Shahzad, Mohsin; Huang, Vincent H.; Qaiser, Tanveer A.; Potluri, Prasanth; Mahl, Sarah E.; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-sheqaih, Nada; Newman, William G.; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N.; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B.; Wu, Doris K.; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C.; Ahmed, Zubair M.; Huang, Taosheng; Riazuddin, Saima

    2015-01-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome. PMID:25807530

  6. Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

    PubMed

    Simon, Mariella; Richard, Elodie M; Wang, Xinjian; Shahzad, Mohsin; Huang, Vincent H; Qaiser, Tanveer A; Potluri, Prasanth; Mahl, Sarah E; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-Sheqaih, Nada; Newman, William G; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B; Wu, Doris K; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C; Ahmed, Zubair M; Huang, Taosheng; Riazuddin, Saima

    2015-03-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome. PMID:25807530

  7. Human endomembrane H sup + pump strongly resembles the ATP-synthetase of Archaebacteria

    SciTech Connect

    Suedhof, T.C.; Stone, D.K.; Johnston, P.A.; Xie, Xiaosong ); Fried, V.A. )

    1989-08-01

    Preparations of mammalian H{sup +} pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H{sup +} pump are unknown from animal cells. The authors report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H{sup +} pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H{sup +} gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H{sup +} pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

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

    SciTech Connect

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

    2006-12-01

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

  9. Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit.

    PubMed Central

    Shiba, K; Suzuki, N; Shigesada, K; Namba, Y; Schimmel, P; Noda, T

    1994-01-01

    We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exceptionally large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain of two repeats of an approximately 90-aa sequence appended to its C-terminal end. While extracts of Escherichia coli do not aminoacrylate mammalian tRNA with isoleucine, expression of the cloned human gene in E. coli results in charging of the mammalian tRNA substrate. The appended third domain is dispensable for detection of this aminoacylation activity and may be needed for assembly of a multisynthetase complex in mammalian cells. Alignment of the sequences of the remaining two domains shared by isoleucyl-tRNA synthetases from E. coli to human reveals a much greater selective pressure on the domain needed for tRNA acceptor helix interactions and catalysis than on the domain needed for interactions with the anticodon. This result may have implications for the historical development of an operational RNA code for amino acids. Images PMID:8052601

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    Nichols, R.C.; Pai, S.I.; Liu, P.; Ge, Q.; Targoff, I.N.

    1995-11-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Jaiswal, Manish; Tétreault, Martine; Donti, Taraka; Sasarman, Florin; Bernard, Geneviève; Demers-Lamarche, Julie; Dicaire, Marie-Josée; Mathieu, Jean; Vanasse, Michel; Bouchard, Jean-Pierre; Rioux, Marie-France; Lourenco, Charles M.; Li, Zhihong; Haueter, Claire; Shoubridge, Eric A.; Graham, Brett H.; Brais, Bernard; Bellen, Hugo J.

    2012-01-01

    An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants. PMID:22448145

  14. Repression of Biotin-Related Proteins by Benzo[a]Pyrene-Induced Epigenetic Modifications in Human Bronchial Epithelial Cells.

    PubMed

    Xia, Bo; Yang, Lin-Qing; Huang, Hai-Yan; Pang, Li; Yang, Xi-Fei; Yi, You-Jin; Ren, Xiao-Hu; Li, Jie; Zhuang, Zhi-Xiong; Liu, Jian-Jun

    2016-05-01

    Benzo[a]pyrene (B[a]P) exposure has been associated with the alteration in epigenetic marks that are involved in cancer development. Biotinidase (BTD) and holocarboxylase synthetase (HCS) are 2 major enzymes involved in maintaining the homeostasis of biotinylation, and the deregulation of this pathway has been associated with a number of cancers. However, the link between B[a]P exposure and the dysregulation of BTD/HCS in B[a]P-associated tumorigenesis is unknown. Here we showed that the expression of both BTD and HCS was significantly decreased upon B[a]P treatment in human bronchial epithelial (16HBE) cells. Benzo[a]pyrene exposure led to the global loss of DNA methylation by immunofluorescence, which coincided with the reduction in acetylation levels on histones H3 and H4 in 16HBE cells. Consistent with decreased histone acetylation, histone deacetylases (HDACs) HDAC2 and HDAC3 were significantly upregulated in a dosage-dependent manner. When DNA methylation or HDAC activity was inhibited, we found that the reduction in BTD and HCS was separately regulated through distinct epigenetic mechanisms. Together, our results suggested the potential link between B[a]P toxicity and deregulation of biotin homeostasis pathway in B[a]P-associated cancer development. PMID:26960346

  15. Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains.

    PubMed

    Lansdon, Eric B; Fisher, Andrew J; Segel, Irwin H

    2004-04-13

    Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the

  16. 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

  17. Expression of gamma-glutamylcysteine synthetase (gamma-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs.

    PubMed Central

    Oguri, T.; Fujiwara, Y.; Isobe, T.; Katoh, O.; Watanabe, H.; Yamakido, M.

    1998-01-01

    We examined the steady-state levels of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) in human lung cancer specimens to elucidate their roles in relation to platinum drug resistance in vivo. Seventy-six autopsy samples (38 primary tumours and their corresponding normal lung tissues) obtained from 38 patients were analysed using the quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Both subunits (heavy and light subunits) of gamma-GCS expression levels of normal lung and tumour tissues exposed to platinum drugs during life were significantly higher than those of non-exposed tissues, whereas only the MRP expression levels of tumours were elevated in association with ante-mortem platinum drug exposure. The gamma-GCS and MRP expression levels correlated significantly. The cMOAT expression levels did not correlate with ante-mortem platinum drug exposure. Next, we monitored gamma-GCS heavy subunit expression levels in peripheral mononuclear cells of eight previously untreated lung cancer patients after platinum drug administration, which revealed that these drugs induced gamma-GCS expression in vivo. These results suggest that gamma-GCS expression is induced by platinum drugs in vivo and/or the physiological stress response to xenobiotics. PMID:9569044

  18. 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.

  19. 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

  20. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in eukaryotic histone H3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BirA ligase, a prokaryotic ortholog of human holocarboxylase synthetase (HCS), is known to biotinylate proteins. Here, we tested the hypothesis that BirA ligase may also catalyze biotinylation of eukaryotic histones. If so, this would render recombinant BirA ligase a useful surrogate for HCS in stud...

  1. 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

  2. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

    PubMed

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

    2016-08-10

    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. PMID:27288593

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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 ...

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

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

    SciTech Connect

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

    2013-11-01

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

  3. 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.

  4. 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

  5. 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

  6. 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

  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. 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

  9. 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 ...

  10. 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

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

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

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

  20. 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.

  1. 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

  2. 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

  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. 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

  5. [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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype

    PubMed Central

    2013-01-01

    Background Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. Methods We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. Results We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. Conclusion This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes. PMID:24103465

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  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. Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.

    PubMed Central

    Ullman, B; Wormsted, M A; Cohen, M B; Martin, D W

    1982-01-01

    Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout. Images PMID:6957854

  12. 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

  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. Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

    PubMed

    Khan, Sameena; Garg, Ankur; Camacho, Noelia; Van Rooyen, Jason; Kumar Pole, Anil; Belrhali, Hassan; Ribas de Pouplana, Lluis; Sharma, Vinay; Sharma, Amit

    2013-05-01

    Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively. PMID:23633587

  15. Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations

    PubMed Central

    Perli, Elena; Fiorillo, Annarita; Giordano, Carla; Pisano, Annalinda; Montanari, Arianna; Grazioli, Paola; Campese, Antonio F.; Di Micco, Patrizio; Tuppen, Helen A.; Genovese, Ilaria; Poser, Elena; Preziuso, Carmela; Taylor, Robert W.; Morea, Veronica; Colotti, Gianni; d'Amati, Giulia

    2016-01-01

    Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNALeu(UUR) or with mutations in the mt-tRNAIle, both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNALys, aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNALeu(UUR) and mt-tRNALys, and stabilize mutant mt-tRNALeu(UUR). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs. PMID:26721932

  16. 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...

  17. 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.

  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, 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Isolation, structure and expression of mammalian genes for histidyl-tRNA synthetase.

    PubMed Central

    Tsui, F W; Siminovitch, L

    1987-01-01

    A full length cDNA clone that codes for human histidyl-tRNA synthetase (HRS) and cDNA clones that span the full length transcript of hamster HRS have been isolated. The full length human HRS cDNA was expressed after transfection into Cos 1 cells and a CHO ts mutant defective in the gene for HRS. The complete nucleotide sequence of the hamster and human gene were obtained and extensive homologies were observed in three regions on comparing these sequences between themselves and with the sequence of HRS derived from yeast. These results provide unequivocal evidence that we have indeed cloned the hamster and human gene for HRS. Three overlapping phage recombinants containing the complete hamster chromosomal gene for HRS have also been isolated. The genomic HRS is divided into 13 exons. The precise locations of each of the 5' and 3' exon-intron boundaries were defined by sequencing the appropriate regions of the cloned genomic DNA and aligning them with the sequence of HRS cDNAs. These studies provide the basis for future structural and functional analysis of the gene for HRS. In particular, it will be of interest to examine if different exons of HRS correlate to different domains of the HRS polypeptide. Images PMID:3554142

  9. 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

  10. 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

  11. Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.

    PubMed Central

    Lamour, V; Quevillon, S; Diriong, S; N'Guyen, V C; Lipinski, M; Mirande, M

    1994-01-01

    An important step ensuring the fidelity in protein biosynthesis is the aminoacylation of tRNAs by aminoacyl-tRNA synthetases. The accuracy of this process rests on a family of 20 enzymes, one for each amino acid. One exception is the formation of Gln-tRNA(Gln) that can be accomplished by two different pathways: aminoacylation of tRNA(Gln) with Gln by glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) or transamidation of Glu from Glu-tRNA(Gln) mischarged by glutamyl-tRNA synthetase (GluRS; EC 6.1.1.17). The latter pathway is widespread among bacteria and organelles that, accordingly, lack GlnRS. However, some bacterial species, such as Escherichia coli, do possess a GlnRS activity, which is responsible for Gln-tRNA(Gln) formation. In the cytoplasm of eukaryotic cells, both GluRS and GlnRS activities can be detected. To gain more insight into the evolutionary relationship between GluRS and GlnRS enzyme species, we have now isolated and characterized a human cDNA encoding GlnRS. The deduced amino acid sequence shows a strong similarity with other known GlnRSs and with eukaryotic GluRSs. A molecular phylogenetic analysis was conducted on the 14 GlxRS (GluRS or GlnRS) sequences available to date. Our data suggest that bacterial GlnRS has a eukaryotic origin and was acquired by a mechanism of horizontal gene transfer. Images PMID:8078941

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

    PubMed Central

    Kim, Jun Seok; Lee, Cheolju

    2015-01-01

    Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

  3. 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

  4. 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)

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

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

    PubMed

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

    2016-05-01

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

  14. Brugia malayi Asparaginyl - tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis

    PubMed Central

    D, Jeeva Jothi; Dhanraj, Muthu; Solaiappan, Shanmugam; Sivanesan, Sanjana; Kron, Michael; Dhanasekaran, Anuradha

    2016-01-01

    A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis. PMID:26751209

  15. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites. PMID:26771178

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

    PubMed Central

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

    2013-01-01

    New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of UMP and dTMP pools thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII deficient mutants restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within or proximal to the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development. PMID:18992249

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

    PubMed

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

    2009-04-01

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

  18. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    PubMed

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites. PMID:26771178

  19. 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

  20. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    PubMed

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes. PMID:25543026

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

    SciTech Connect

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

    2012-07-11

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

  2. 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

  3. 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

  4. Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase

    SciTech Connect

    Greco, A.; Gong, S.S.; Ittmann, M.; Basilico, C. . School of Medicine)

    1989-06-01

    The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. The authors have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G+C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far they have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.

  5. Investigating Arsenic Susceptibility from a Genetic Perspective in Drosophila Reveals a Key Role for Glutathione Synthetase

    PubMed Central

    Muñiz Ortiz, Jorge G.; Opoka, Robert; Kane, Daniel; Cartwright, Iain L.

    2009-01-01

    Chronic exposure to arsenic-contaminated drinking water can lead to a variety of serious pathological outcomes. However, differential responsiveness within human populations suggests that interindividual genetic variation plays an important role. We are using Drosophila to study toxic metal response pathways because of unrivalled access to varied genetic approaches and significant demonstrable overlap with many aspects of mammalian physiology and disease phenotypes. Genetic analysis (via chromosomal segregation and microsatellite marker-based recombination) of various wild-type strains exhibiting relative susceptibility or tolerance to the lethal toxic effects of arsenite identified a limited X-chromosomal region (16D-F) able to confer a differential response phenotype. Using an FRT-based recombination approach, we created lines harboring small, overlapping deficiencies within this region and found that relative arsenite sensitivity arose when the dose of the glutathione synthetase (GS) gene (located at 16F1) was reduced by half. Knockdown of GS expression by RNA interference (RNAi) in cultured S2 cells led to enhanced arsenite sensitivity, while GS RNAi applied to intact organisms dramatically reduced the concentration of food-borne arsenite compatible with successful growth and development. Our analyses, initially guided by observations on naturally occurring variants, provide genetic proof that an optimally functioning two-step glutathione (GSH) biosynthetic pathway is required in vivo for a robust defense against arsenite; the enzymatic implications of this are discussed in the context of GSH supply and demand under arsenite-induced stress. Given an identical pathway for human GSH biosynthesis, we suggest that polymorphisms in GSH biosynthetic genes may be an important contributor to differential arsenic sensitivity and exposure risk in human populations. PMID:18779381

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. The phenotypic expression of mitochondrial tRNA-mutations can be modulated by either mitochondrial leucyl-tRNA synthetase or the C-terminal domain thereof

    PubMed Central

    Giordano, Carla; Morea, Veronica; Perli, Elena; d’Amati, Giulia

    2015-01-01

    Mutations in mitochondrial (mt) DNA determine important human diseases. The majority of the known pathogenic mutations are located in transfer RNA (tRNA) genes and are responsible for a wide range of currently untreatable disorders. Experimental evidence both in yeast and in human cells has shown that the detrimental effects of mt-tRNA point mutations can be attenuated by increasing the expression of the cognate mt-aminoacyl-tRNA synthetases (aaRSs). In addition, constitutive high levels of isoleucyl-tRNA syntethase have been shown to reduce the penetrance of a homoplasmic mutation in mt-tRNAIle in a small kindred. More recently, we showed that the isolated carboxy-terminal domain of human mt-leucyl tRNA synthetase (LeuRS-Cterm) localizes to mitochondria and ameliorates the energetic defect in transmitochondrial cybrids carrying mutations either in the cognate mt-tRNALeu(UUR) or in the non-cognate mt-tRNAIle gene. Since the mt-LeuRS-Cterm does not possess catalytic activity, its rescuing ability is most likely mediated by a chaperon-like effect, consisting in the stabilization of the tRNA structure altered by the mutation. All together, these observations open potential therapeutic options for mt-tRNA mutations-associated diseases. PMID:25852750

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

    SciTech Connect

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

    2015-10-20

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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Calpain Cleaves Most Components in the Multiple Aminoacyl-tRNA Synthetase Complex and Affects Their Functions*

    PubMed Central

    Lei, Hui-Yan; Zhou, Xiao-Long; Ruan, Zhi-Rong; Sun, Wei-Cheng; Eriani, Gilbert; Wang, En-Duo

    2015-01-01

    Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNAGln and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca2+ and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC. PMID:26324710

  16. Calpain Cleaves Most Components in the Multiple Aminoacyl-tRNA Synthetase Complex and Affects Their Functions.

    PubMed

    Lei, Hui-Yan; Zhou, Xiao-Long; Ruan, Zhi-Rong; Sun, Wei-Cheng; Eriani, Gilbert; Wang, En-Duo

    2015-10-23

    Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNA(Gln) and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca(2+) and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC. PMID:26324710

  17. Structure of Leishmania major Methionyl-tRNA Synthetase in Complex with Intermediate Products Methionyladenylate and Pyrophosphate

    PubMed Central

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

    2011-01-01

    Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg2+ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of E. coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNAMet complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme. PMID:21144880

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

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

    PubMed

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

    2016-01-01

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

  8. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

    PubMed

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-02-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically. PMID:26836257

  9. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

    PubMed Central

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-01-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically. PMID:26836257

  10. Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides.

    PubMed

    Bao, Y B; Li, L; Ye, M X; Dong, Y H; Jin, W X; Lin, Z H

    2013-01-01

    The blood cockle, Tegillarca granosa, is a widely consumed clam in the Indo-Pacific region. Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. We identified the GS of T. granosa (Tg-GS) from hemocytes by 3'- and 5'-rapid amplification of cDNA ends (RACE)-PCR. The full-length cDNA consisted of 1762 bp, with a 1104-bp open reading frame encoding 367 amino acids. Sequence comparison showed that Tg-GS has homology to GS of other organisms, with 79.78% identity with GS from the Pacific oyster Crassostrea gigas, 71.98% identity with GS from the zebrafish Danio rerio, and 68.96% identity with human Homo sapiens GS. A C-beta-Grasp domain and an N-catalytic domain were identified in Tg-GS, indicating that Tg-GS should be classified as a new member of the GS family. A quantitative RT-PCR assay was used to detect mRNA expression of Tg-GS in five different tissues. Higher levels of mRNA expression of GS were detected in the tissues of hemocytes and the mantle. Up-regulation of GS by challenge with the bacteria Vibrio parahaemolyticus and with bacterial wall lipopolysaccharides showed that GS plays a role in anti-bacterial immunity. We conclude that pathogen infection significantly induces expression level of Tg- GS, and that activation of GS influences the immune response of T. granosa by increasing glutamine concentration. PMID:23661439