Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pyrococcus furiosus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 523 records · Page 29Linked to original sources
PubMed1996

General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid.

Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two beta-galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O-nitrophenyl beta-D-galactopyranoside as substrate.

Anti-Bacterial Agents↗
PubMed2006

Identity elements required for enzymatic formation of N2,N2-dimethylguanosine from N2-monomethylated derivative and its possible role in avoiding alternative conformations in archaeal tRNA.

Here, we have investigated the specificity of purified recombinant tRNA:m(2)(2)G10 methyltransferase of Pyrococcus abyssi ((Pab)Trm-m(2)(2)G10 enzyme). This archaeal enzyme catalyses mono- and dimethylation of the N(2)-exocyclic amino group of guanine at position 10 of several tRNA species. Our results indicate that only few identity elements are required for the efficient formation of m(2)(2)G10. They are composed of a G10.U25 wobble base-pair in the dihydrouridine arm (D-arm) and a four nucleotide variable loop (V-loop) within a canonical three-dimensional (3D) structure. The types of base-pairs in the D-arm or amino acid acceptor stem are also important for the enzymatic reaction, but appear to affect only the rate of tRNA methylation. However, in tRNA species harbouring a G10-C25 Watson-Crick base-pair and/or five nucleotide V-loop, only m(2)G10 is produced. To impair the monomethylation reaction, drastic amputation in the T-arm is required. Our observations contrast with those reported earlier for the identity elements required for a remotely related Pyrococcus furiosus Trm-m(2)(2)G26 enzyme (alias (Pfu)Trm1) that also catalyses the two step formation of m(2)(2)G but at position 26 in several tRNA species. In this case, a G10-C25 base-pair together with the five nucleotide V-loop were shown to be required for efficient formation of m(2)(2)G26. Thus, in the Pyrococcus genus, the major identity elements that preclude formation of m(2)(2)G at positions 10 or 26 in tRNA are mutually exclusive. Therefore, the Trm-m(2)(2)G10 and Trm-m(2)(2)G26 enzymes have evolved independently towards different specificities. In addition, identity elements for m(2)/m(2)(2)G10 formation in archaeal tRNA are different from the ones required for m(2)G10 formation in eukaryal tRNA. We propose that archaeal tRNA:m(2)(2)G10 methyltransferases, unlike the orthologous eukaryal tRNA:m(2)G10 methyltransferases, evolved towards m(2)(2)G10 specificity due to the possible requirement of preventing formation of alternative structures in G/C rich archaeal tRNA species.

Archaeal Proteins↗
PubMed1999

Anaerobic microbes: oxygen detoxification without superoxide dismutase.

Superoxide reductase from the hyperthermophilic anaerobe Pyrococcus furiosus uses electrons from reduced nicotinamide adenine dinucleotide phosphate, by way of rubredoxin and an oxidoreductase, to reduce superoxide to hydrogen peroxide, which is then reduced to water by peroxidases. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and therefore confers a selective advantage on anaerobes. Superoxide reductase and associated proteins are catalytically active 80 degrees C below the optimum growth temperature (100 degrees C) of P. furiosus, conditions under which the organism is likely to be exposed to oxygen.

Acetylation↗
PubMed1998

Novel disulfide oxidoreductase in search of a function.

The high-resolution structure of an oxidoreductase from the archaeon Pyrococcus furiosus shows that it comprises two adjacent domains each with the thioredoxin (trx/grx) fold. Its functional properties are not yet fully defined but it may be related to the multi-domain eukaryotic protein disulfide-isomerases.

Binding Sites↗
PubMed1998

A novel DNA polymerase family found in Archaea.

One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical alpha-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one alpha-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499-512, 1997). The genes in M. jannaschii encoding the proteins that are homologous to the DNA polymerase II of P. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia coli had both DNA polymerizing and 3'-->5' exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

Amino Acid Sequence↗
PubMed1999

Quantitative analysis of pyroglutamic acid in peptides.

A simplified and rapid procedure for the determination of pyroglutamic acid in peptides was developed. The method involves the enzymatic cleavage of an N-terminal pyroglutamate residue using a thermostable pyroglutamate aminopeptidase and isocratic HPLC separation of the resulting enzymatic hydrolysate using a column switching technique. Pyroglutamate aminopeptidase from a thermophilic archaebacteria, Pyrococcus furiosus, cleaves N-terminal pyroglutamic acid residue independent of the molecular weight of the substrate. It cleaves more than 85% of pyroglutamate from peptides whose molecular weight ranges from 362.4 to 4599.4 Da. Thus, a new method is presented that quantitatively estimates N-terminal pyroglutamic acid residue in peptides.

Amphibian Proteins↗
PubMed2001

Cloning, expression, and structure analysis of carbamate kinase-like carbamoyl phosphate synthetase from Pyrococcus abyssi.

Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100 degrees C. Carbamoyl phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form carbamoyl phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.

Amino Acid Sequence↗
PubMed1996

In vitro hydrogen production by glucose dehydrogenase and hydrogenase.

A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity.

Cloning, Molecular↗
PubMed1997

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

Amino Acid Sequence↗
PubMed1997

Stability and dynamics in a hyperthermophilic protein with melting temperature close to 200 degrees C.

The rubredoxin protein from the hyperthermophilic archaebacterium Pyrococcus furiosus was examined by a hydrogen exchange method. Even though the protein does not exhibit reversible thermal unfolding, one can determine its stability parameters-free energy, enthalpy, entropy, and melting temperature-and also the distribution of stability throughout the protein, by using hydrogen exchange to measure the reversible cycling of the protein between native and unfolded states that occurs even under native conditions.

Circular Dichroism↗
PubMed1998

A heterodimeric DNA polymerase: evidence that members of Euryarchaeota possess a distinct DNA polymerase.

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase delta, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.

Amino Acid Sequence↗
PubMed2005

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion.

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.

Adenosine Triphosphatases↗
PubMed1997

Shuttle vectors for hyperthermophilic archaea.

Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea.

Alcohol Dehydrogenase↗
PubMed2005

Cloning, expression, and purification of the general stress protein YhbO from Escherichia coli.

We cloned, expressed, and purified the Escherichia coli yhbO gene product, which is an amino acid sequence homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21 (DE3) E. coli strain transformed with the YhbO-expression vector, pET-21a-yhbO, accumulates large amounts of a soluble protein with a molecular mass of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by ion exchange chromatography and hydroxyapatite chromatography, and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric, and hexameric forms. We also report a strong sequence homology between YhbO and the general stress protein YfkM (64% identities), which suggests that YhbO is a stress protein, and a strong structural homology between YhbO and the Pyrococcus horikoshii intracellular protease PhpI. We could not, however, detect any proteolytic or peptidolytic activity of YhbO, using classical biochemical substrates.

Chromatography, Liquid↗
PubMed1999

Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: purification and characterization of key enzymes.

Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60 degrees C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enzyme.

Amino Acid Sequence↗
PubMed2000

Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution.

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

Amino Acid Sequence↗
PubMed1998

Sequence of archaeal Methanococcus jannaschii alpha-amylase contains features of families 13 and 57 of glycosyl hydrolases: a trace of their common ancestor?

Two sequentially different, seemingly unrelated alpha-amylase families exist, known as family-13 and family-57 glycosyl hydrolases. Despite the common enzyme activity, it has as yet been impossible to detect any sequence similarity between the two families. The detailed analysis of the recently determined sequence of the alpha-amylase from methanogenic archaeon Methanococcus jannaschii using the sensitive Hydrophobic Cluster Analysis method revealed that this alpha-amylase contains features of both families of alpha-amylases. Thus the M. jannaschii alpha-amylase is similar to the Pyrococcus furiosus alpha-amylase from family 57 while it obviously contains most of the sequence fingerprints characteristic for alpha-amylase family 13. Importantly, a glutamic acid residue equivalent with the family-13 catalytic glutamate positioned in the beta 5-strand segment was identified in members of family 57. The results presented in this report indicate that the two families, 13 and 57, are either the products of a very distant common ancestor or have evolved from each other, although at present they can represent two different alpha-amylase families with evolved different catalytic mechanisms, catalytic machinery and folds.

Amino Acid Sequence↗
PubMed1998

Cloning and nucleotide sequence of the thermostable beta-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme.

Pyrococcus woesei (DSM 3773) beta-galactosidase gene amplified by polymerase chain reaction was cloned into KpnI and HindIII binding sites of pET-30LIC expression plasmid. The obtained pGal2 (6785 bp) transcription vector was then transferred to Escherichia coli B121 (DE3) cells. High identity (99.9%) of DNA sequences suggests that beta-galactosidases from P. woesei and Pyrococcus furiosus are closely related. This enzyme from E. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85 degrees C. The crude beta-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted from E. coli cells and has maximal activity at pH 5.4 and temperature of 93 degrees C. Isolated enzyme is active at temperatures up to 110 degrees C and the activity loss after 4 h of incubation at 85 and 93 degrees C did not exceed 11 and 15% of the initial value respectively.

Amino Acid Sequence↗