In vitro phosphorylation of initiation factor 2 alpha (aIF2 alpha) from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric protein composed of alpha, beta, and gamma subunits, of which the alpha subunit (eIF2 alpha) plays a crucial role in regulation of protein synthesis through phosphorylation at Ser51. All three subunit genes are conserved in Archaea. To examine the properties of archaeal initiation factor 2 alpha (aIF2 alpha), three genes encoding alpha, beta, and gamma subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2 alpha, aIF2 beta, and aIF2 gamma, were characterized with reference to the properties of eIF2. aIF2 alpha preferentially interacts with aIF2 gamma, but does not interact with aIF2 beta, which is consistent with data obtained with eIF2, of which eIF2 gamma serves as a core subunit, interacting with eIF2 alpha and eIF2 beta. It was found that aIF2 alpha was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) from human, and a primary target site was suggested to be Ser48 within aIF2 alpha. This finding led us to the search for a putative aIF2 specific kinase gene (PH0512) in the P. horikoshii genome. The gene product Ph0512p unambiguously phosphorylated aIF2 alpha, and Ser48, as in the phosphorylation by hPKR, was suggested to be a target amino acid residue for the PKR homologue Ph0152p in P. horikoshii. These findings suggest that aIF2 alpha, like eIF2 alpha in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.     

Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5'-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5'-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95 degrees C and retained at least half the activity after 9 min at 95 degrees C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of kcat, Km, DNA, and Km, AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.     

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi. Insights into cooperative and allosteric mechanisms

Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature, the transition between T- and R-states involves significant changes in the quaternary structure, which were detected by analytical ultracentrifugation. The enzyme is allosterically regulated by ATP (activator) and by CTP and UTP (inhibitors). Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas ATP and UTP act at millimolar concentrations, CTP inhibits at micromolar concentrations, suggesting that in P. abyssi, inhibition by CTP is the major control of enzyme activity. While V(max) increased with temperature, cooperative and allosteric effects were little or not affected, showing that molecular adaptation to high temperature allows the flexibility required to form the appropriate networks of interactions. In contrast to the same enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the carbamyl phosphate analogue phosphonacetate; this difference supports the idea that in native cells ATCase interacts with carbamyl phosphate synthetase to channel the highly thermolabile carbamyl phosphate.     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.     

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi: thermostability and 1.8A resolution crystal structure of the catalytic subunit complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate

The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits. The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells. At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable. Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C. Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability. To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli. Interactions with PALA are strictly conserved. This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit. Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini. Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.     

Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii

Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N-terminal domain (NTD) of PriL (PriL(NTD); residues 1-222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at 2.9 A resolution. The PriL(NTD) structure consists of two subdomains, the helix-bundle and twisted-strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull-down and surface plasmon resonance analyses of structure-based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155-Tyr156-Ile157 region near the flexible region is the PriS-binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000-fold. These findings and a structural comparison with a previously reported PriL(NTD)-PriS complex suggest that the presented alternative conformations of the twisted-strand domain facilitate the heterodimer assembly.     

Structural switch of the gamma subunit in an archaeal aIF2 alpha gamma heterodimer

Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. This study reports the crystallographic structure of an aIF2alphagamma heterodimer from Sulfolobus solfataricus bound to Gpp(NH)p-Mg(2+). aIF2gamma is in a closed conformation with the G domain packed on domains II and III. The C-terminal domain of aIF2alpha interacts with domain II of aIF2gamma. Conformations of the two switch regions involved in GTP binding are similar to those encountered in an EF1A:GTP:Phe-tRNA(Phe) complex. Comparison with the EF1A structure suggests that only the gamma subunit of the aIF2alphagamma heterodimer contacts tRNA. Because the alpha subunit markedly reinforces the affinity of tRNA for the gamma subunit, a contribution of the alpha subunit to the switch movements observed in the gamma structure is considered.     

Three-dimensional structure of methionyl-tRNA synthetase from Pyrococcus abyssi

In class 1 aminoacyl-tRNA synthetases, methionyl-tRNA synthetases (MetRS) are homodimers or monomers depending on the presence or absence of a domain appended at the C-side of the polypeptide chain. Beyond this C-domain, all MetRS display a highly conserved catalytic core with a Rossmann fold, the two halves of which are linked by a connective peptide (CP). Three-dimensional folding of CP and its putative zinc content have served as a basis to propose a division of the MetRS family into four subgroups. All subgroups but one, which is predicted to display two zincs per MetRS polypeptide, have been characterized. In the present study, the 3D structure of MetRS from Pyrococcus abyssi could be solved at 2.9 A resolution. The data obtained and atomic absorption spectroscopic measurements establish the presence of two metal ions per polypeptide chain. This finding brings strong support to the above classification. In the crystal, the C-terminal dimerization domain is disordered. This observation is thought to reflect marked flexibility of the two core moieties with respect to the C-domains in the dimer. Gel shift experiments were performed with the isolated C-terminal dimerization domain and a core monomeric MetRS, both derived from the P. abyssi enzyme. Complex formation between the C-domain and the core enzyme could not be evidenced. Moreover, association of tRNA(Met) to the core enzyme is enhanced in the presence of the C-domain. Together, these experiments suggest positive control in trans by the C-domain on recognition of tRNA by the core moiety of MetRS.     

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles.     

Construction of a shuttle vector for,and spheroplast transformation of,the hyperthermophilic archaeon Pyrococcus abyssi

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.     

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.     

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.     

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli.

The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.     

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (~1.6 MDa) complex that is not active. The apparent K_m values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 10⁴ mM⁻¹ · s⁻¹ and 0.39 mM and 3.40 × 10³ mM⁻¹ · s⁻¹, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k_cat/K_m = 32 (mM · s)⁻¹]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (~5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.     

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.     

A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli , and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.