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PubMed1998

Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep-sea hydrothermal ecosystem.

A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria: Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl-protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and P'1 positions. Its activity is optimal at 95 degrees C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.

Amino Acid Sequence↗
PubMed1999

Channeling of carbamoyl phosphate to the pyrimidine and arginine biosynthetic pathways in the deep sea hyperthermophilic archaeon Pyrococcus abyssi.

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

Adaptation, Biological↗
PubMed1995

Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents.

A minimal growth medium containing only nine amino acids and vitamins as the sole carbon and energy sources allowed the growth of Pyrococcus abyssi GE 5, a novel hyperthermophilic sulfur-metabolizing archaeon isolated from deep-sea hydrothermal vents. The generation time in this medium was about 40 min, and cell densities up to 5 x 10(sup8) cells ml(sup-1) were attained. These results are similar to those obtained previously with complex proteinaceous media.

Journal Article↗
PubMed2004

Pyrococcus abyssi alkaline phosphatase: the dimer is the active form.

Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.

Alkaline Phosphatase↗
PubMed2000

Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms.

Recent work suggests that restriction-modification gene complexes are mobile genetic elements that insert themselves into the genome and cause various genome rearrangements. In the present work, the complete genome sequences of Pyrococcus horikoshii and Pyrococcus abyssi, two species in a genus of hyperthermophilic archaeon (archaebacterium), were compared to detect large genome polymorphisms linked with restriction-modification gene homologs. Sequence alignments, GC content analysis, and codon usage analysis demonstrated the diversity of these homologs and revealed a possible case of relatively recent acquisition (horizontal transfer). In two cases out of the six large polymorphisms identified, there was insertion of a DNA segment with a modification gene homolog, accompanied by target deletion (simple substitution). In two other cases, homologous DNA segments carrying a modification gene homolog were present at different locations in the two genomes (transposition). In both cases, substitution (insertion/deletion) in one of the two loci was accompanied by inversion of adjacent chromosomal segment. In the fifth case, substitution by a DNA segment carrying type I restriction, modification, and specificity gene homologs was likewise accompanied by adjacent inversion. In the last case, two homologous DNA segments, were found at different loci in the two genomes (transposition), but only one of them had insertion of a modification homolog and an unknown ORF. The possible relationship of these polymorphisms to attack by restriction enzymes on the chromosome will be discussed.

Chromosome Inversion↗
PubMed2002

Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

Adenosine Triphosphatases↗
PubMed2002

Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya.

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.

Amino Acid Sequence↗
PubMed2003

Identification, purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi.

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mm. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.

Amino Acid Sequence↗
PubMed2005

Cloning, purification and crystallization of a Walker-type Pyrococcus abyssi ATPase family member.

Several ATPase proteins play essential roles in the initiation of chromosomal DNA replication in archaea. Walker-type ATPases are defined by their conserved Walker A and B motifs, which are associated with nucleotide binding and ATP hydrolysis. A family of 28 ATPase proteins with non-canonical Walker A sequences has been identified by a bioinformatics study of comparative genomics in Pyrococcus genomes. A high-throughput structural study on P. abyssi has been started in order to establish the structure of these proteins. 16 genes have been cloned and characterized. Six out of the seven soluble constructs were purified in Escherichia coli and one of them, PABY2304, has been crystallized. X-ray diffraction data were collected from selenomethionine-derivative crystals using synchrotron radiation. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 79.41, b = 48.63, c = 108.77 A, and diffract to beyond 2.6 A resolution.

Adenosine Triphosphatases↗
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↗
PubMed2003

Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex.

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.

Amino Acid Sequence↗
PubMed2000

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Base Sequence↗
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

Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.

The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, some of which catalyze unique conversions. Moreover, the enzymes appear not to be regulated allosterically, but rather at transcriptional level. To elucidate details of the gene expression control, the transcription initiation sites of the glycolytic genes in Pyrococcus furiosus have been mapped by primer extension analysis and the obtained promoter sequences have been compared with upstream regions of non-glycolytic genes. Apart from consensus sequences for the general transcription factors (TATA-box and BRE) this analysis revealed the presence of a potential transcription factor binding site (TATCAC-N(5)-GTGATA) in glycolytic and starch utilizing promoters of P. furiosus and several thermococcal species. The absence of this inverted repeat in Pyrococcus abyssi and Pyrococcus horikoshii probably reflects that their reduced catabolic capacity does not require this regulatory system. Moreover, this phyletic pattern revealed a TrmB-like regulator (PF0124 and TK1769) which may be involved in recognizing the repeat. This Thermococcales glycolytic regulon, with more than 20 genes, is the largest regulon that has yet been described for Archaea.

Base Sequence↗
PubMed1996

UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains.

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi.

Archaea↗