SEARCH · PubMed Health

Results for “Pyrococcus abyssi”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed2002

Holliday junction-resolving enzymes from eight hyperthermophilic archaea differ in reactions with cruciform DNA.

Holliday junction-resolving enzymes have been identified in a broad variety of organisms and tissues. In this study, six new Holliday junction-cleaving enzymes (Hjcs) were obtained from hyperthermophilic crenarchaeal and euryarchaeal species, including Pyrococcus horikoshii, Pyrococcus abyssi, Methanococcus jannaschii, Methanobacterium thermautotrophicum, Archaeoglobus fulgidus, and Aeropyrum pernix. The genes were cloned and overexpressed in Escherichia coli, and the respective proteins were purified from crude extracts to homogeneity. For an initial characterization of the enzymatic activities, synthetic heat-stable fixed and mobile cruciform DNA substrates were used at 75 degrees C. The Hjcs from Pyrococcus furiosus, Sulfolobus solfataricus, and the archaeal virus SIRV2 were included in the study for comparison. Despite their sequence homology, the enzymes showed marked differences in their reactions with individual cruciform DNAs. While the fixed cruciform structure was cleaved by all enzymes at only one major position, the mobile cruciform structure displayed different cleavage patterns for individual Hjcs, each with several cleavage positions. Furthermore, a strong bias for cleavage of one direction across the junction was observed with the fixed cruciform DNA for all enzymes. In contrast, the mobile cruciform DNA displayed different preferences, depending on the enzyme used.

Amino Acid Sequence↗

PubMed2004

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic investigations of a unique editing domain from archaebacteria.

Threonyl-tRNA synthetase (ThrRS) faces a crucial double-discrimination problem during the translation of genetic code. Most ThrRSs from the archaeal kingdom possess a unique editing domain that differs from those of eubacteria and eukaryotes. In order to understand the structural basis of the editing mechanism in archaea, the editing module of ThrRS from Pyrococcus abyssi comprising of the first 183 amino-acid residues was cloned, expressed, purified and crystallized. The crystals belong to the trigonal space group P3(1(2))21, with one molecule in the asymmetric unit.

Cloning, Molecular↗

PubMed2000

DbClustal: rapid and reliable global multiple alignments of protein sequences detected by database searches.

DbClustal addresses the important problem of the automatic multiple alignment of the top scoring full-length sequences detected by a database homology search. By combining the advantages of both local and global alignment algorithms into a single system, DbClustal is able to provide accurate global alignments of highly divergent, complex sequence sets. Local alignment information is incorporated into a ClustalW global alignment in the form of a list of anchor points between pairs of sequences. The method is demonstrated using anchors supplied by the Blast post-processing program, Ballast. The rapidity and reliability of DbClustal have been demonstrated using the recently annotated Pyrococcus abyssi proteome where the number of alignments with totally misaligned sequences was reduced from 20% to <2%. A web site has been implemented proposing BlastP database searches with automatic alignment of the top hits by DbClustal.

Algorithms↗

PubMed2003

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

DNA↗

PubMed2004

N2-methylation of guanosine at position 10 in tRNA is catalyzed by a THUMP domain-containing, S-adenosylmethionine-dependent methyltransferase, conserved in Archaea and Eukaryota.

In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed (Pab)Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N(2)) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N(2)-monomethyl (m(2)G) or N(2)-dimethylguanosine (m(2)(2)G). Interestingly, (Pab)Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ((Pfu)Trm-G26, also known as (Pfu)Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.

Amino Acid Sequence↗

PubMed2000

Carbamate kinase can replace in vivo carbamoyl phosphate synthetase. Implications for the evolution of carbamoyl phosphate biosynthesis.

The exclusive involvement of carbamate kinase (CK) in fermentative ATP production and of carbamoyl phosphate synthetase (CPS) in the production of carbamoyl phosphate (CP) for pyrimidines and arginine biosynthesis was challenged by the finding of CK as the only activity synthesising CP in the archaea Pyrococcus furiosus and Pyrococcus abyssi. We now show that CK can replace CPS in vivo: transformation of Escherichia coli devoid of the CPS gene with plasmids encoding the CK from P. furiosus or from Enterococcus faecalis (which uses CK for making ATP) restores the ability of CPS-deficient E. coli to grow in the absence of arginine and uracil if ammonia and bicarbonate are present.

Ammonia↗

PubMed2001

The intein of the Thermoplasma A-ATPase A subunit: structure, evolution and expression in E. coli.

BACKGROUND: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. RESULTS: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain.T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. CONCLUSIONS: Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

Adenosine Triphosphatases↗

PubMed2005

Expression, purification, crystallization and preliminary crystallographic analysis of the PAB0955 gene product.

PAB0955 from Pyrococcus abyssi is a prototype of a new Walker-type ATPase/GTPase conserved in archaea and eukaryota but not found in bacteria. PAB0955 has been expressed, purified and crystallized, and it has been shown that this thermostable protein is dimeric in reductive conditions. Crystals have been obtained either without nucleotide or in the presence of GDP or GTPgammaS. Preliminary X-ray crystallographic data up to 2.08 A resolution have been collected from these crystals.

Acid Anhydride Hydrolases↗

PubMed2001

In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin.

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.

Base Sequence↗

PubMed2002

Structure and function of the C-terminal domain of methionyl-tRNA synthetase.

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.

Archaeal Proteins↗

PubMed1997

Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales.

A collection of 57 strains of hyperthermophilic Archaea from the order Thermococcales was screened for the presence of plasmids; 9 plasmids present in six of these strains were isolated and characterized in terms of size and cross-hybridization. The Notl macrorestriction patterns of genomic DNA of strains harbouring these plasmids were obtained. Pyrococcus abyssi strains GE27 and GE23 as well as Thermococcus sp. GE31 contained a single plasmid of 3.5 kb (pGN27), 16.8 kb (pGN23) and 5.3 kb (pGN31), respectively, whilst the three strains I559, I560 and I690 all contained two plasmids of 3.5 kb (pSN559, pSN560, pSN690) and 24 kb (pLN559, pLN560, pLN690), respectively. Plasmid pGN27 strongly cross-hybridized with the previously described plasmid pGT5 from P. abyssi strain GE5, whilst plasmids pGN23 and pGN31 did not cross-hybridize with each other, nor with any other plasmid. The three small plasmids of strains I559, I560 and I690 cross-hybridized, as well as their three large plasmids. Macrorestriction pattern analysis and the results of plasmid cross-hybridization experiments indicated that these three strains were different but closely related, and likely belonged to the genus Thermococcus. This study shows that plasmids are widespread in hyperthermophilic archaea, and significantly increases the number and diversity of plasmids available for laboratory work.

Blotting, Southern↗

PubMed2001

In vitro replication slippage by DNA polymerases from thermophilic organisms.

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.

Artifacts↗

PubMed2003

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism.

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

Amino Acid Sequence↗

PubMed2002

The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors.

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Amino Acid Sequence↗

PubMed2000

Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75.

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topo-isomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.

Amino Acid Sequence↗

PubMed2001

Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp.

Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Base Sequence↗

PubMed1999

The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.

Adenosine Diphosphate↗

PubMed1999

The active site of the rolling circle replication protein Rep75 is involved in site-specific nuclease, ligase and nucleotidyl transferase activities.

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via a rolling circle mechanism. The protein Rep75, encoded by this plasmid, exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin sequence. In addition, Rep75 catalyses a site-specific nucleotidyl terminal transferase (NTT) activity, e.g. it can transfer one AMP or dAMP (from ATP or dATP) to the 3'-OH of an oligonucleotide corresponding to the left part of the nicking site. The Rep75 sequence contains a motif similar to the active-site motifs of Rep proteins from the PhiX174/pC194 superfamily. We show here that the tyrosine present in this motif is indeed essential for DNA cleavage by Rep75, but is dispensable for its NTT activity. However, a nearby arginine, which is not required for DNA cleavage, is involved in both NTT and closing, indicating that the same active site is involved in the NC and NTT activities of Rep75. For both NTT and NC, the G residue in 3' of the nicking site is essential, whereas the A residue in 5' is dispensable for NC, despite its conservation in RC plasmids of the PhiX174/pC194 superfamily. The NTT and closing activities have an optimal temperature lower than the nicking activity. These data indicate that the three reactions catalysed by Rep75 can be uncoupled, although they share part of their mechanisms. Finally, we show that NC is inhibited by ATP or dATP at concentrations that promote NTT. We propose a model in which the NTT activity of Rep75 plays a role in the regulation of pGT5 replication in vivo.

Amino Acid Sequence↗