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Merike Kelve - One of the best experts on this subject based on the ideXlab platform.
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expression and characterization of recombinant 2 5 oligoadenylate Synthetase from the marine sponge geodia cydonium
FEBS Journal, 2007Co-Authors: Mailis Pari, Anne Kuusksalu, Merike Kelve, Annika Lopp, Tonu Reintamm, Just JustesenAbstract:2',5'-oligoadenylate (2-5A) Synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A Synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A Synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A Synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A Synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A Synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A Synthetase/bacterial RNA complex activity.
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2 5 oligoadenylate Synthetase from a lower invertebrate the marine sponge geodia cydonium does not need dsrna for its enzymatic activity
Biochimica et Biophysica Acta, 2002Co-Authors: Annika Lopp, Anne Kuusksalu, Werner E G Muller, Tonu Reintamm, Merike KelveAbstract:Recently, the presence of 2',5'-linked oligoadenylates and a high 2',5'-oligoadenylate Synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2-5A Synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2-5A Synthetases, the 2-5A Synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2-5A Synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells. These results indicate that the 2-5A Synthetase from G. cydonium may be active per se or is activated by some other mechanism. The sponge enzyme is capable of synthesizing a series of 2-5A oligomers ranging from dimers to octamers. The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates. By its product profile and kinetics of formation, the sponge 2-5A Synthetase behaves like a specific isoform of enzymes of the 2-5A Synthetase family.
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origin of the interferon inducible 2 5 oligoadenylate Synthetases cloning of the 2 5 oligoadenylate Synthetase from the marine sponge geodia cydonium
FEBS Letters, 1999Co-Authors: Matthias Wiens, Anne Kuusksalu, Merike Kelve, Werner E G MullerAbstract:In vertebrates cytokines mediate innate (natural) immunity and protect them against viral infections. The cytokine interferon causes the induction of the (2′-5′)oligoadenylate Synthetase [(2-5)A Synthetase], whose product, (2′-5′)oligoadenylate, activates the endoribonuclease L which in turn degrades (viral) RNA. Three isoforms of (2-5)A Synthetases exist, form I (40–46 kDa), form II (69 kDa), and form III (100 kDa). Until now (2-5)A Synthetases have only been cloned from birds and mammals. Here we describe the cloning of the first putative invertebrate (2-5)A Synthetase from the marine sponge Geodia cydonium. The deduced amino acid sequence shows signatures characteristic for (2-5)A Synthetases of form I. Phylogenetic analysis of the putative sponge (2-5)A Synthetase indicates that it diverged first from a common ancestor of the hitherto known members of (vertebrate) (2-5)A Synthetases I, (2-5)A Synthetases II and III. Moreover, it is suggested that the (2-5)A Synthetases II and III evolved from this common ancestor (very likely) by gene duplication. Together with earlier results on the existence of the (2′-5′)oligoadenylates in G. cydonium, the data presented here demonstrate that also invertebrates, here sponges, are provided with the (2-5)A system. At present, it is assumed that this system might be involved in growth control, including control of apoptosis, and acquired its additional function in innate immune response in evolutionarily younger animals, in vertebrates.
Paul Schimmel - One of the best experts on this subject based on the ideXlab platform.
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orthogonal use of a human trna Synthetase active site to achieve multifunctionality
Nature Structural & Molecular Biology, 2010Co-Authors: Quansheng Zhou, Min Guo, Mili Kapoor, Rajesh Belani, William B Kiosses, Melanie Hanan, Chulho Park, Eva R Armour, Leslie A Nangle, Paul SchimmelAbstract:Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA Synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA Synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the Synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the Synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA Synthetases and other proteins.
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species specific microhelix aminoacylation by a eukaryotic pathogen trna Synthetase dependent on a single base pair
Biochemistry, 1995Co-Authors: Cheryl L Quinn, Nianjun Tao, Paul SchimmelAbstract:We report here that tyrosyl-tRNA Synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA Synthetase and aligns with the prokaryotic tyrosyl-tRNA Synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region. The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr). Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices. However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair. We show that P. carinii tyrosyl-tRNA Synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr. In contrast, the enzyme does not charge E. coli microhelixTyr. Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P. carinii tyrosyl-tRNA Synthetase. Conversely, we found that E. coli tyrosyl-tRNA Synthetase can charge an E. coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair. The results demonstrate that the common structural framework of homologous tRNA Synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation. We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA Synthetases.
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functional dissection of a predicted class defining motif in a class ii trna Synthetase of unknown structure
Biochemistry, 1994Co-Authors: Matthew W Davis, Douglas D Buechter, Paul SchimmelAbstract:A core of eight beta-strands and three alpha-helices was recently predicted for the active site domain of Escherichia coli alanyl-tRNA Synthetase, an enzyme of unknown structure [Ribas de Pouplana, L1., Buechter, D. D., Davis, M. W., & Schimmel, P. (1993) Protein Sci. 2, 2259-2262; Shi, J.-P., Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 26, 5312-5318]. A critical part of this predicted structure is two antiparallel beta-strands and an intervening loop that make up the second of three highly degenerate sequence motifs that are characteristic of the class II aminoacyl-tRNA Synthetases. We present here an in vivo and in vitro analysis of 21 rationally designed mutations in the predicted 34-amino acid motif 2 of E. coli alanyl-tRNA Synthetase. Although this motif in E. coli alanyl-tRNA Synthetase is of a different size than and has only two sequence identities with the analogous motif in yeast aspartyl- and Thermus thermophilus seryl-tRNA Synthetases, whose structures are known, the functional consequences of the mutations are explainable in terms of those structures. In particular, the analysis demonstrates the importance of the predicted motif 2 in adenylate formation, distinguishes between two similar, but distinct, predicted models for this motif, and distinguishes between the functional importance of two adjacent phenylalanines in a way that strongly supports the predicted structure. The results suggest that similar analyses will be generally useful in testing models for active site regions of other class II aminoacyl-tRNA Synthetases of unknown structure.
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an operational rna code for amino acids and possible relationship to genetic code
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Paul Schimmel, Richard Giegé, Dino Moras, Shigeyuki YokoyamaAbstract:RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA Synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA Synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial Synthetase, which was needed for expression of the operational RNA code. The second Synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of Synthetases was added with the anticodon-containing domain of tRNAs.
Dino Moras - One of the best experts on this subject based on the ideXlab platform.
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transfer rna mediated editing in threonyl trna Synthetase the class ii solution to the double discrimination problem
Cell, 2000Co-Authors: Annecatherine Dockbregeon, Rajan Sankaranarayanan, Pascale Romby, Joel Caillet, Mathias Springer, B Rees, Christopher S Francklyn, Chantal Ehresmann, Dino MorasAbstract:Threonyl-tRNA Synthetase, a class II Synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of Synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of Synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.
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the free yeast aspartyl trna Synthetase differs from the trna asp complexed enzyme by structural changes in the catalytic site hinge region and anticodon binding domain
Journal of Molecular Biology, 2000Co-Authors: Claude Sauter, Dino Moras, Bernard Lorber, Jean Cavarelli, Richard GiegéAbstract:Abstract Aminoacyl-tRNA Synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its Synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound Synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA Synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNAAsp. The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the Synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the Synthetase anticodon binding module.
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the first step of aminoacylation at the atomic level in histidyl trna Synthetase
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: John G Arnez, Dino Moras, John G Augustine, Christopher S FrancklynAbstract:The crystal structure of an enzyme–substrate complex with histidyl-tRNA Synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA Synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA Synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA Synthetases.
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an operational rna code for amino acids and possible relationship to genetic code
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Paul Schimmel, Richard Giegé, Dino Moras, Shigeyuki YokoyamaAbstract:RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA Synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA Synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial Synthetase, which was needed for expression of the operational RNA code. The second Synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of Synthetases was added with the anticodon-containing domain of tRNAs.
Kenneth Stuart - One of the best experts on this subject based on the ideXlab platform.
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a spectrophotometric assay for quantitative measurement of aminoacyl trna Synthetase activity
Journal of Biomolecular Screening, 2013Co-Authors: Igor Cestari, Kenneth StuartAbstract:Aminoacyl-tRNA Synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high-throughput assay for measuring aminoacyl-tRNA Synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA Synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA Synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product and yielded a Z′ factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA Synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 ...
Richard Giegé - One of the best experts on this subject based on the ideXlab platform.
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the free yeast aspartyl trna Synthetase differs from the trna asp complexed enzyme by structural changes in the catalytic site hinge region and anticodon binding domain
Journal of Molecular Biology, 2000Co-Authors: Claude Sauter, Dino Moras, Bernard Lorber, Jean Cavarelli, Richard GiegéAbstract:Abstract Aminoacyl-tRNA Synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its Synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound Synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA Synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNAAsp. The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the Synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the Synthetase anticodon binding module.
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a domain in the n terminal extension of class iib eukaryotic aminoacyl trna Synthetases is important for trna binding
The EMBO Journal, 2000Co-Authors: Magali Frugier, Luc Moulinier, Richard GiegéAbstract:Cytoplasmic aspartyl-tRNA Synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20–70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA Synthetases. By gel retardation studies and footprinting experiments on yeast tRNAAsp, we show that the extension, connected to the anticodon-binding module of the Synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb Synthetases identifies a lysine-rich 11 residue sequence (29LSKKALKKLQK39 in yeast AspRS with the consensus xSKxxLKKxxK in class IIb Synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.
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an operational rna code for amino acids and possible relationship to genetic code
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Paul Schimmel, Richard Giegé, Dino Moras, Shigeyuki YokoyamaAbstract:RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA Synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA Synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial Synthetase, which was needed for expression of the operational RNA code. The second Synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of Synthetases was added with the anticodon-containing domain of tRNAs.
