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At least 667 records · Page 37Linked to original sources
PubMed1997

Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp. KOD1.

The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcussp. KOD1, was expressed in Escherichia coli. The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form. A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity. The reaction product of this DNase activity was mononucleotides. The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively. In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity. The protein exhibited no DNase activity in the presence of Zn2+ion, which was one of the most preferable divalent cations for ATPase activity. Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP.Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity.

Adenosine Triphosphatases↗
PubMed1990

The hyperthermophilic archaebacterium, Pyrococcus furiosus. Development of culturing protocols, perspectives on scaleup, and potential applications.

From this brief discussion, it is clear that there are many obstacles to overcome before hyperthermophilic archaebacteria will be an important aspect of biotechnology. Nevertheless, the prospects are intriguing. The nature of the environments that harbor these organisms and the consequent requirements for their controlled culture suggest that chemical and biochemical engineers can play an important role in elucidating their scientific and technological aspects.

Archaea↗
PubMed2001

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea.

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Archaeal Proteins↗
PubMed1996

Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

Archaea↗
PubMed2006

Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate.

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.

Amino Acid Sequence↗
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↗
PubMed2000

Evidence of recent lateral gene transfer among hyperthermophilic archaea.

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Amino Acid Sequence↗
PubMed2006

Sources, properties and suitability of new thermostable enzymes in food processing.

Investigations concerning recombinant a-amylases from Pyrococcus woesei and thermostable a-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant ss-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are also presented. The activity of this enzyme in a wide pH range of 4.3-6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant a-amylase and ss-galactosidase was facilitated by cloning and expression of genes from Pyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained a-amylase has maximal activity at pH 5.6 and 93 degrees C. The half-life of this preparation (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. An advantageous attribute of recombinant a -amylase is independence of its activity and stability on calcium salt. a-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8-6.9. Thus, this enzyme can be used together with recombinant a-amylase.

Animals↗