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Paola Londei - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of the translation factor aif2 5b in the Thermophilic Archaeon sulfolobus solfataricus
Molecular Microbiology, 2007Co-Authors: Enzo Maone, Dario Benelli, Michele Di Stefano, Alessandra Berardi, Stefano Marzi, Anna La Teana, Paola LondeiAbstract:The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of ‘chimeric’ proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the Thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.
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Functional analysis of the translation factor aIF2/5B in the Thermophilic Archaeon Sulfolobus solfataricus
Molecular Microbiology, 2007Co-Authors: Enzo Maone, Dario Benelli, Michele Di Stefano, Alessandra Berardi, Stefano Marzi, Anna La Teana, Paola LondeiAbstract:The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of ‘chimeric’ proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the Thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.
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single molecule imaging by atomic force microscopy of the native chaperonin complex of the Thermophilic Archaeon sulfolobus solfataricus
Biochemical and Biophysical Research Communications, 2001Co-Authors: Francesco Valle, Giovanni Dietler, Paola LondeiAbstract:Abstract The chaperonin of the extremely Thermophilic Archaeon Sulfolobus solfataricus has been imaged for the first time under native conditions using the atomic force microscope. This technique allows to visualize the structure of biomolecules in solution under physiological conditions providing a nanometer resolution topographic image of the sample. Single molecule studies can reveal fine structural details, providing a powerful insight into the active conformation of a macromolecule, and also allowing to detect different conformational states corresponding to functional changes.
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in vitro processing of the 16s rrna of the Thermophilic Archaeon sulfolobus solfataricus
Journal of Bacteriology, 2001Co-Authors: Andrea Ciammaruconi, Paola LondeiAbstract:In both bacteria and eukaryotes, rRNAs are synthesized as large precursors which are processed to mature rRNA species. However, the maturation pathways, and the enzymatic machinery involved, differ in the two cell domains (for reviews, see references 7 and 9). The principal maturation enzyme in bacteria is RNase III, which cleaves within the long double-stranded stems formed by the inverted repeats flanking both large rRNA genes, releasing precursor 16S and 23S rRNAs, which are subsequently trimmed at both ends to yield the mature molecules. However, RNase III is not strictly essential, and mutants lacking this enzymatic activity are viable (although slow growing) because there exist alternative processing pathways for producing mature rRNAs, especially the 16S rRNA (9). In eukaryotes, the rRNA genes are not flanked by inverted repeats and there are no processing stems: rRNA maturation is performed by ribonucleoprotein enzymes that cut at specific sites within the transcribed spacers. The compositions and mechanisms of action of these enzymes are still largely unknown, although it is well established that they require the presence of several small nucleolar RNAs such as U3, U8, U14, and others (7, 25). However, eukaryotes also possess an RNase III homolog that in Saccharomyces cerevisiae has been shown to be involved in rRNA processing both in vitro and in vivo (1). In contrast with the wealth of data available for the other two primary domains, our knowledge of rRNA processing in archaea is still very fragmentary. As in bacteria, the archaeal large rRNA genes are flanked by imperfect inverted repeats that pair, forming long double-helical stems. These stems are truncated by an enzyme which probably recognizes a specific structure, a bulge-helix-bulge (BHB) motif (4, 8, 13, 15). A peculiar situation seems to exist in the crenArchaeon Sulfolobus acidocaldarius, where 16S rRNA maturation was reported to be independent of the formation of the processing stem (6), thus resembling the early steps of eukaryotic 18S RNA maturation. In this paper we discuss the in vitro processing of 16S rRNA in the extremely Thermophilic Archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and various protein preparations. We show that the 5′ external transcribed spacer (5′ETS) of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also present in the early A0 and 0 processing sites of yeast and vertebrates. This site is probably specific to the Crenarchaeota, as it is recognized and cleaved by heterologous cell extracts from this archaeal branch only. Furthermore, S. solfataricus pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S sequence, in correspondence with the BHB motif. The endonuclease responsible for this cleavage is present in cell extracts from both Crenarchaeota and Euryarchaeota. The processing sites were the same regardless of whether the substrate was the naked RNA or a ribonucleoprotein particle. Under no experimental conditions was maturation of either the 5′ or the 3′ end of the 16S RNA observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.
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cis acting signals controlling translational initiation in the Thermophilic Archaeon sulfolobus solfataricus
Molecular Microbiology, 1999Co-Authors: Ivano Condo, Paola Londei, Andrea Ciammaruconi, Dario Benelli, Davide RuggeroAbstract:In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely Thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis-acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine-Dalgarno (SD)-like ribosome-binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5' untranslated region altogether, thereby creating a 'leaderless' mRNA. This suggests that 'leaderless' initiation operates by a default mechanism that does not require a specific mRNA-rRNA interaction and may be common to all three primary domains of life.
Wolfram Zillig - One of the best experts on this subject based on the ideXlab platform.
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relationships between fuselloviruses infecting the extremely Thermophilic Archaeon sulfolobus ssv1 and ssv2
Research in Microbiology, 2003Co-Authors: Kenneth M Stedman, Hans Peter Arnold, Ingelore Holz, Hien Phan, Roger A Garrett, Wolfram ZilligAbstract:The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.
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The Genome of the Archaeal Virus SIRV1 Has Features in Common with Genomes of Eukaryal Viruses
Virology, 2001Co-Authors: Helmut Blum, Wolfram Zillig, S. Mallok, Horst Domdey, David PrangishviliAbstract:Abstract The virus SIRV1 of the extremely Thermophilic Archaeon Sulfolobus has a double-stranded DNA genome similar in architecture to the genomes of eukaryal viruses of the families Poxviridae, Pycodnaviridae, and Asfarviridae: the two strands of the 32,301 bp long linear genome are covalently connected forming a continuous polynucleotide chain and 2029 kb long inverted repeats are present at the termini. Very likely it also shares with these viruses mechanisms of initiation of replication and resolution of replicative intermediates.
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viruses of the extremely Thermophilic Archaeon sulfolobus
Trends in Microbiology, 2001Co-Authors: David Prangishvili, Kenneth M Stedman, Wolfram ZilligAbstract:Abstract Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus–host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.
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genetic elements in the extremely Thermophilic Archaeon sulfolobus
Extremophiles, 1998Co-Authors: Wolfram Zillig, David Prangishvili, Kenneth M Stedman, Hans Peter Arnold, Ingelore Holz, Anja Schweier, Hien Phan, Roger A Garrett, Jakob K KristjanssonAbstract:This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons.
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bioenergetics and cytoplasmic membrane stability of the extremely acidophilic Thermophilic Archaeon picrophilus oshimae
Extremophiles, 1998Co-Authors: J L C M Van De Vossenberg, Arnold J M Driessen, Wolfram Zillig, W N KoningsAbstract:Picrophilus oshimae is an extremely acidophilic, Thermophilic Archaeon that grows optimally at 60°C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane.
Christa Schleper - One of the best experts on this subject based on the ideXlab platform.
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candidatus nitrosocaldus cavascurensis an ammonia oxidizing extremely Thermophilic Archaeon with a highly mobile genome
Frontiers in Microbiology, 2018Co-Authors: Sophie S Abby, Michael Melcher, Melina Kerou, Mart Krupovic, Michaela Stieglmeier, Claudia Rossel, Kevin Pfeifer, Christa SchleperAbstract:Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a Thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68 • C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.
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candidatus nitrosocaldus cavascurensis an ammonia oxidizing extremely Thermophilic Archaeon with a highly mobile genome
bioRxiv, 2017Co-Authors: Sophie S Abby, Michael Melcher, Melina Kerou, Mart Krupovic, Michaela Stieglmeier, Claudia Rossel, Kevin Pfeifer, Christa SchleperAbstract:Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first cultivated Thermophilic representative with a sequenced genome, which allows to search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been cultivated from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 25h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase, genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.
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an insertion element of the extremely Thermophilic Archaeon sulfolobus solfataricus transposes into the endogenous β galactosidase gene
Molecular Genetics and Genomics, 1994Co-Authors: Christa Schleper, Richard Roder, Tatjana Singer, Wolfram ZilligAbstract:Three phenotypically stable mutants of the extremely Thermophilic Archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.
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Nucleotide sequence, transcription and phylogeny of the gene encoding the superoxide dismutase of Sulfolobus acidocaldarius
Biochimica et Biophysica Acta, 1993Co-Authors: Hans-peter Klenk, Christa Schleper, Volker Schwass, Ronald BrudlerAbstract:Abstract The gene encoding the superoxide dismutase (SOD) of the Thermophilic Archaeon Sulfolobus acidocaldarius has been isolated and sequenced. Both the start site and the termination sites of the corresponding transcript were mapped. The deduced amino acid sequence of the protein is very similar to the sequence of mangenese- or iron-containing SODs. Phylogenetic sequence analysis corroborated the monophyletic nature of the archaeal domain.
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the particle ssv1 from the extremely Thermophilic Archaeon sulfolobus is a virus demonstration of infectivity and of transfection with viral dna
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Christa Schleper, K Kubo, Wolfram ZilligAbstract:Abstract The lemon-shaped "virus-like" particle SSV1 produced by the Thermophilic Archaeon Sulfolobus shibatae has not previously been observed to infect any host. Using a plaque assay suitable for the extreme growth conditions of this Archaeon, we have shown infection of Sulfolobus solfataricus by SSV1. Upon infection, the viral genome was always found integrated into a tRNA gene of the host chromosome, a situation similar to that in S. shibatae, proving that site-specific integration is involved in establishing the lysogenic state. As in S. shibatae, UV-irradiation of lysogenized S. solfataricus led to virus production apparently not accompanied by cell lysis. We have also demonstrated the efficient uptake of exogenous DNA and its expression in Sulfolobus by transfecting S. solfataricus with SSV1 DNA by electroporation. Transfection efficiencies of up to 10(6) transfectants per microgram of DNA were obtained.
Michael J Danson - One of the best experts on this subject based on the ideXlab platform.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase multienzyme complex in the Thermophilic Archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W Hough, Michael J DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the Thermophilic Archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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Stepwise Adaptations of Citrate Synthase to Survival at Life'S Extremes. From Psychrophile to Hyperthermophile.
FEBS Journal, 2002Co-Authors: Graeme S. Bell, David W Hough, Michael J Danson, Rupert J. Russell, Helen Connaris, Garry L. TaylorAbstract:The crystal structure of citrate synthase from the Thermophilic Archaeon Sulfolobus solfataricus (optimum growth temperature ¼ 85 � C) has been determined, extending the number of crystal structures of citrate synthase from different organisms to a total of five that span the temperature range over which life exists (from psychrophile to hyperthermophile). Detailed structural analysis has revealed possible molecular mechanisms that determine the different stabilities of the five proteins. The key to these mechanisms is the precise structural location of the additional interactions. As one ascends the temperature ladder, the subunit interface of this dimeric enzyme and loop regions are reinforced by complexelectrostatic interactions, and there is a reduced exposure of hydrophobic surface. These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and interfacial regions.
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kinetics and mechanism of the citrate synthase from the Thermophilic Archaeon thermoplasma acidophilum
Biochemistry, 2000Co-Authors: Linda C Kurz, Rupert J. M. Russell, George R Drysdale, Marian Riley, Maharaj Alejandro Tomar, Judy Chen, Michael J DansonAbstract:The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis...
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Citrate synthase from the hyperThermophilic Archaeon, Pyrococcus furiosus
Protein Engineering, 1995Co-Authors: Jacqueline M. Muir, David W Hough, Rupert J. M. Russell, Michael J DansonAbstract:The gene encoding the enzyme citrate synthase has been cloned and sequenced from the hyperThermophilic Archaeon Pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with citrate synthases from archaeal, bacterial and eukaryal organisms. The gene has been over-expressed in Escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties. A structurally-based sequence alignment was made to the citrate synthase from the Thermophilic Archaeon Thermoplasma acidophilum, the crystal structure of which we have determined recently. From this alignment, a homology-modelled structure for the P.furiosus citrate synthase was generated and analysed.
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the crystal structure of citrate synthase from the Thermophilic Archaeon thermoplasma acidophilum
Structure, 1994Co-Authors: Rupert J. M. Russell, David W Hough, Michael J Danson, G L TaylorAbstract:Abstract Background: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the Thermophilic Archaea tolerate temperatures in the range 55–110°C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15–45°C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. Results We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). Conclusion The three-dimensional structural comparison of Thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, Thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.
Jennifer A Littlechild - One of the best experts on this subject based on the ideXlab platform.
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biochemical and structural studies of a l haloacid dehalogenase from the Thermophilic Archaeon sulfolobus tokodaii
Extremophiles, 2009Co-Authors: M N Isupov, Andrey Lebedev, Jennifer A LittlechildAbstract:Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the Thermophilic Archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.
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An order–disorder twin crystal of l‐2‐haloacid dehalogenase from Sulfolobus tokodaii
Acta Crystallographica Section D-biological Crystallography, 2007Co-Authors: Michail N. Isupov, Andrey Lebedev, Jennifer A LittlechildAbstract:The l-2-haloacid dehalogenase enzymes catalyse the hydrolytic cleavage of a halogen from the C2 position of short-chain haloacids. The recombinant dehalogenase from the Thermophilic Archaeon Sulfolobus tokodaii has been cloned, overexpressed and purified to homogeneity. The 24 kDa enzyme was crystallized using the microbatch method in the monoclinic space group C2, with unit-cell parameters a = 127.6, b = 58.1, c = 51.2 A, β = 97.2°. Data were collected to 1.9 A resolution using synchrotron radiation and the structure was solved by molecular replacement. Analysis of the data and the preliminary refined model showed that the crystal was an order–disorder twin by reticular merohedry with a twin index of 10. It was possible to detwin the experimental data utilizing the symmetry of the molecular layers from which the crystal is built.
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An order-disorder twin crystal of L-2-haloacid dehalogenase from Sulfolobus tokodaii.
Acta crystallographica. Section D Biological crystallography, 2007Co-Authors: Michail N. Isupov, Andrey A Lebedev, Jennifer A LittlechildAbstract:The L-2-haloacid dehalogenase enzymes catalyse the hydrolytic cleavage of a halogen from the C2 position of short-chain haloacids. The recombinant dehalogenase from the Thermophilic Archaeon Sulfolobus tokodaii has been cloned, overexpressed and purified to homogeneity. The 24 kDa enzyme was crystallized using the microbatch method in the monoclinic space group C2, with unit-cell parameters a = 127.6, b = 58.1, c = 51.2 A, beta = 97.2 degrees . Data were collected to 1.9 A resolution using synchrotron radiation and the structure was solved by molecular replacement. Analysis of the data and the preliminary refined model showed that the crystal was an order-disorder twin by reticular merohedry with a twin index of 10. It was possible to de-twin the experimental data utilizing the symmetry of the molecular layers from which the crystal is built.
