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PubMed2006

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus Pyrococcus from an Algerian hydrothermal hot spring.

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80-85 degrees C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.

Algeria↗
PubMed2006

Survival and growth of two heterotrophic hydrothermal vent archaea, Pyrococcus strain GB-D and Thermococcus fumicolans, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes.

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5-7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature.

Hot Temperature↗
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↗
PubMed1999

Thermostable aminopeptidase from Pyrococcus horikoshii.

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.

Aminopeptidases↗
PubMed1998

Isolation of a minD-like gene in the hyperthermophilic archaeon Pyrococcus AL585, and phylogenetic characterization of related proteins in the three domains of life.

The region upstream of the dinF-like gene of the hyperthermophilic archaeon Pyrococcus strain AL585 has been cloned and sequenced. This region contains an open reading frame (ORF) that encodes a polypeptide with a high similarity to MinD proteins and their Mrp paralogues. Transcripts of the dinF-like and the minD-like genes were detected by RT-PCR, indicating that they are both expressed in vivo. The MinD and MinD-like proteins belong to a broad family of ATPases involved in chromosome and plasmid partitioning. MinD-like proteins can be defined by specific amino-acid sequence signatures. A systematic search for proteins sharing these signatures in current databases and newly sequenced genomes show that MinD-like proteins are present in all archaeal genomes sequenced so far, often in several copies. Phylogenetic analysis identifies two groups of MinD-like proteins which are also characterized by more conserved amino-acid motifs. A first group, which includes the Escherichia coli MinD and the Pyrococcus AL585 MinDL protein, contains only procaryotic proteins. This group can be further divided into a subgroup of archaeal proteins and two subgroups of bacterial proteins. A second group includes proteins more related to the E. coli Mrp protein and contains representants of the three domains of life. The conservation of MinD-like proteins in the three domains of life suggests that these proteins play a central role in cellular metabolism.

Adenosine Triphosphatases↗
PubMed2003

Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR.

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.

Archaeal Proteins↗
PubMed2002

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

Amino Acid Sequence↗
PubMed2002

Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc.

The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular alpha-amylase that belongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaeal homologues (e.g., Bacillus licheniformis TAKA-term and Pyrococcus kodakaraensis KOD1 alpha-amylases, respectively) even without adding Ca(2+) ions. Unlike the TAKA-therm amylase that contains no cysteine, the P. furiosus enzyme contains five cysteines (C152, C153, C165, C387, and C430), only four of which (C152, C153, C387, and C430) are conserved in the P. kodakaraensis alpha-amylase. To test the potential function of cysteines in P. furiosus alpha-amylase stability, these five residues were substituted with Ser or Ala-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramatically destabilized P. furiosus alpha-amylase. At the same time, the quadruple mutant enzyme C152S/C153S/C387S/C430A (mutant SSCSA) was as thermostable as the wild-type enzyme. Mutant SSCSA and wild-type alpha-amylases were strongly destabilized by dithiothreitol and ethylenediaminetetraacetic acid, suggesting that metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomic emission spectrometry showed the presence of Ca(2+) and Zn(2+) metal ions in P. furiosus alpha-amylase. Although Ca(2+) is known to contribute to alpha-amylase's stability, the absence of two out of the three conserved Ca(2+) ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca(2+) binding. We also provide evidence suggesting that Cys165 is involved in Zn(2+) binding and that Cys165 is essential for the stability of P. furiosus alpha-amylase at very high temperatures.

Amino Acid Substitution↗
PubMed2004

Theoretical model of the three-dimensional structure of a sugar-binding protein from Pyrococcus horikoshii: structural analysis and sugar-binding simulations.

The three-dimensional structure of a sugar-binding protein from the thermophilic archaea Pyrococcus horikoshii has been predicted by a homology modelling procedure and investigated for its stability and its ability to bind different sugars. The model was created by using as templates the three-dimensional structures of a maltodextrin-binding protein from Pyrococcus furiosus, a trehalose-maltose-binding protein from Thermococcus litoralis and a maltodextrin-binding protein from Escherichia coli. According to the suggestions from the CASP (Critical Assessment of Structure Prediction) meetings, the homology modelling strategy was applied by assessing an accurate multiple sequence alignment, based on the high structural conservation in the family of ATP-binding cassette transporters to which all these proteins belong. The model has been deposited in the Protein Data Bank with the code 1R25. According to the origin of the protein, several characteristics in the organization of the secondary-structure elements and in the distribution of polar and non-polar amino acids are very similar to those of thermophilic proteins, compared with proteins from mesophilic organisms, and are analysed in detail. Finally, a simulation of the binding of several sugars in the binding site of this protein is presented, and interactions with amino acids are highlighted in detail.

Amino Acid Sequence↗
PubMed2006

Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.

Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

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↗
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↗
PubMed2001

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Amino Acid Sequence↗
PubMed2003

Structural evidence for a hydride transfer mechanism of catalysis in phosphoglucose isomerase from Pyrococcus furiosus.

In the Euryarchaeota species Pyrococcus furiosus and Thermococcus litoralis, phosphoglucose isomerase (PGI) activity is catalyzed by an enzyme unrelated to the well known family of PGI enzymes found in prokaryotes, eukaryotes, and some archaea. We have determined the crystal structure of PGI from Pyrococcus furiosus in native form and in complex with two active site ligands, 5-phosphoarabinonate and gluconate 6-phosphate. In these structures, the metal ion, which in vivo is presumed to be Fe2+, is located in the core of the cupin fold and is immediately adjacent to the C1-C2 region of the ligands, suggesting that Fe2+ is involved in catalysis rather than serving a structural role. The active site contains a glutamate residue that contacts the substrate, but, because it is also coordinated to the metal ion, it is highly unlikely to mediate proton transfer in a cis-enediol mechanism. Consequently, we propose a hydride shift mechanism of catalysis. In this mechanism, Fe2+ is responsible for proton transfer between O1 and O2, and the hydride shift between C1 and C2 is favored by a markedly hydrophobic environment in the active site. The absence of any obvious enzymatic machinery for catalyzing ring opening of the sugar substrates suggests that pyrococcal PGI has a preference for straight chain substrates and that metabolism in extreme thermophiles may use sugars in both ring and straight chain forms.

Binding Sites↗
PubMed2001

Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus.

The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

Amino Acid Sequence↗
PubMed1999

Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription.

Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.

Archaeal Proteins↗
PubMed2005

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus.

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.

Amino Acid Sequence↗
PubMed2002

A Pyrococcus homolog of the leucine-responsive regulatory protein, LrpA, inhibits transcription by abrogating RNA polymerase recruitment.

The genomes of Archaea harbor homologs of the global bacterial regulator leucine-responsive regulatory protein (Lrp). Archaeal Lrp homologs are helix-turn-helix DNA-binding proteins that specifically repress the transcription of their own genes in vitro. Here, we analyze the interaction of Pyrococcus LrpA with components of the archaeal transcriptional machinery at the lrpA promoter. DNA-protein complexes can be isolated by electrophoretic mobility shift assays that contain both LrpA and the two archaeal transcription factors TBP and TFB. Phenanthroline-copper footprinting experiments showed that the DNA-binding sites of LrpA and TBP/TFB do not overlap. These results and the finding that association of RNA polymerase with the TBP-TFB promoter complex was inhibited in the presence of LrpA indicate that Pyrococcus LrpA interferes with RNA polymerase recruitment. A DNA motif required for the LrpA-DNA interaction was inferred from dimethylsulfate methylation interference experiments.

Archaeal Proteins↗