Pyrococcus Kodakaraensis KOD1 Research Articles

Structural Mechanism for Coordination of Proofreading and Polymerase Activities in Archaeal DNA Polymerases

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.

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.

Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an “opened” conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A β-hairpin motif (residues 242–249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.

20] Methylguanine methyltransferase from Thermococcus kodakaraensis KOD1

Enzymes derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD 1 (previously reported as Pyrococcus kodakaraensis KOD1) have been shown to be extremely thermostable, and amino acid sequences of those enzymes are closely related to eukaryal homologs. This chapter describes the purification and biochemical characterization of the O 6 -meG DNA methyltransferases (MGMT) from T. kodakaraensis KOD1 (Tk-MGMT), the amino acid sequence of which exhibits homology with similar enzymes from mammals, yeast, and bacteria. Hyperthermophilic archaea live in an environment that is very similar to that of the primitive earth. Early organisms must have been exposed to deleterious, high-temperature environments. It was therefore essential to develop effective DNA repair systems. Hence, the study of archaeal MGMT is expected to provide interesting clues concerning primitive systems of DNA repair and their high thermostability. The detailed structural analysis is now in progress using the crystals mentioned above. Three-dimensional structure allows to elucidate the relationship between structure and function of their enzymes.

Acceptor specificity of 4-α-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose

4-α-Glucanotransferase from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 showed a broad acceptor specificity to various saccharides in an intermolecular transglycosylation reaction. In particular, the enzyme produced large amounts of transfer products of various acceptors such as d-glucose, methyl-α- d-glucoside, phenyl-α- d-glucoside, and d-xylose. It is suggested that the requirement for an effective acceptor in the intermolecular transglycosylation reaction catalyzed by this enzyme is the pyranose structure with the same configurations of the free C2-, C3-, and C4-hydroxyl groups as d-glucopyranose, like cyclomaltodextrin glucanotransferase (CGTase). However, the enzyme showed some acceptor specificities unlike those of CGTase. Analysis of the action of 4-α-glucanotransferase indicated that the enzyme catalyzes an intramolecular trans-glycosylation (cyclization) reaction of amylose to produce cyclic α-1,4-glucan (cycloamylose). The yield of cycloamylose reached 67%, and the degree of polymerization was found to range from 16 to above 55.

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 ( Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80°C. However, the protein showed little GTPase activity at 40°C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [α- 32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42°C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90°C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.

Effect of polyamines on histone-induced DNA compaction of hyperthermophilic archaea

The effect of polyamines on histone-mediated DNA compaction was examined in vitro with archaeal histone HpkA from Pyrococcus kodakaraensis KOD1. An agarose gel mobility-shift experiment indicated that histone-bound DNA (compacted DNA) was further compacted by addition of a polyamine (putrescine, spermidine, or spermine) or its acetylated form ( N-acetylputrescine, N 1-acetylspermidine, N 8-acetylspermidine, or N 1-acetylspermine) when the mixture was incubated at above 75°C. Spermine was most effective in compaction enhancement among all the polyamines tested. A high concentration of potassium ion (1.0 M) did not stabilize the compacted form of DNA even though double-stranded DNA was stably maintained against thermal denaturation at elevated temperatures under this condition. It appears likely that multivalent polyamines have a nucleosome maintenance function in hyperthermophilic archaea in high-temperature environments.

Acceptor Specificity of 4-.ALPHA.-Glucanotransferase from Pyrococcus kodakaraensis KOD1, and Synthesis of Cycloamylose.

4-α-Glucanotransferase from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 showed a broad acceptor specificity to various saccharides in an intermolecular transglycosylation reaction. In particular, the enzyme produced large amounts of transfer products of various acceptors such as d -glucose, methyl-α- d -glucoside, phenyl-α- d -glucoside, and d -xylose. It is suggested that the requirement for an effective acceptor in the intermolecular transglycosylation reaction catalyzed by this enzyme is the pyranose structure with the same configurations of the free C2-, C3-, and C4-hydroxyl groups as d -glucopyranose, like cyclomaltodextrin glucanotransferase (CGTase). However, the enzyme showed some acceptor specificities unlike those of CGTase. Analysis of the action of 4-α-glucanotransferase indicated that the enzyme catalyzes an intramolecular trans-glycosylation (cyclization) reaction of amylose to produce cyclic α-1,4-glucan (cycloamylose). The yield of cycloamylose reached 67%, and the degree of polymerization was found to range from 16 to above 55.

The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA.

The entire gene cluster encoding enzymes involved in biosynthesis of L-tryptophan in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 has been cloned and sequenced. Seven ORFs, which encode indole-3-glycerol phosphate synthase (trpC), anthranilate phosphoribosyltransferase (trpD), the two subunits of anthranilate synthase (trpEG), phosphoribosyl anthranilate isomerase (trpF) and the two subunits of tryptophan synthase (trpAB), were identified. The gene order is trpCDEGFBA, covering a region of 6045 bp. In order to confirm the function of the gene products, we expressed the first gene, Pk-trpC, in Escherichia coli. The protein product was purified, and was found to show the expected indole-3-glycerol phosphate synthase activity, with a temperature optimum of 85 degrees C. We could clearly identify a single mRNA transcript by Northern analysis using probes in the central and 3'-regions of the gene cluster, indicating that the gene cluster is transcribed as an operon. A significant increase in trp mRNA level was observed in cells grown in medium depleted of L-tryptophan, compared to cells grown in medium supplemented with L-tryptophan, indicating that expression of the gene cluster is regulated at the transcriptional level.

Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure

We previously reported the presence of a highly active, carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. In this study, structural analysis of Pk-Rubisco has been performed. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk-Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. In order to investigate the existence of small subunits in native Pk-Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk-Rubisco was detected when the cell-free extracts of P. kodakaraensis KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk-Rubiscos exhibited identical mobilities on native-PAGE. These results indicated that native Pk-Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk-Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk-Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 Å resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk-Rubisco estimated from gel filtration, strongly suggest that Pk-Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos.

Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase.

DNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and betaG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG(1), <font face="k">k. K(d) values were 7.3 x 10(-8) for 3G8 and 1.1 x 10(-6) for betaG1. Nucleotide sequencing of cDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among alpha-like (family B) DNA polymerases (Region II), and the epitope for betaG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).

Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA

We have isolated TBP (TATA-binding protein)-interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein ( Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25 558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, K D, of 1.24–1.46 μM. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription.

Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

Five clustered genes ( flaB1, flaB2, flaB3, flaB4 and flaB5) for multiple subunits of flagellar filaments from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were cloned and sequenced. Deduced amino acid sequences were aligned and it was revealed that five flagellin genes are homologous especially in the N-terminal hydrophobic region which might be important for interaction among flagellin subunits. Phylogenetic analysis was performed among archaeal flagellins from methanogens, an extreme halophile and our hyperthermophile, indicating that KOD1 flagellins were grouped together with methanogenic counterparts and were distinguishable from halophilic flagellins. Northern analysis of transcripts from flagellin genes from P. kodakaraensis KOD1 revealed that four major transcripts (0.98, 3.7, 5.4 and 9.2 kb) initiating from immediately upstream of flaB1 encode different combinations of five flagellins.

Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

Five clustered genes (flaB1, flaB2, flaB3, flaB4 and flaB5) for multiple subunits of flagellar filaments from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were cloned and sequenced. Deduced amino acid sequences were aligned and it was revealed that five flagellin genes are homologous especially in the N-terminal hydrophobic region which might be important for interaction among flagellin subunits. Phylogenetic analysis was performed among archaeal flagellins from methanogens, an extreme halophile and our hyperthermophile, indicating that KOD1 flagellins were grouped together with methanogenic counterparts and were distinguishable from halophilic flagellins. Northern analysis of transcripts from flagellin genes from P. kodakaraensis KOD1 revealed that four major transcripts (0.98, 3.7, 5.4 and 9.2 kb) initiating from immediately upstream of flaB1 encode different combinations of five flagellins.

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue ( Pk-REC) from a hyperthermophilic archaeon

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 ( Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

Presence of a Structurally Novel Type Ribulose-bisphosphate Carboxylase/Oxygenase in the Hyperthermophilic Archaeon,Pyrococcus kodakaraensis KOD1

We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P. kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 degreesC, both higher than any previously characterized Rubisco. The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC. Northern blot analysis demonstrated that the gene was transcribed in P. kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P. kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells.

Gene analysis and enzymatic properties of thermostable β-glycosidase from Pyrococcus kodakaraensis KOD1

A β-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding β-glycosidase ( Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other β-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to β-mannosidase from P. furiosus (55.7%), β-glycosidase from Sulfolobus solfataricus (42.7%) and β-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The β-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100°C, and had a half-life of 18 h at 90°C. The β-glycosidase hydrolyzed various pNp-β-glycopyranosides, with K cat K m values in the order of pNp-β-glucopyranoside≒pNp-β-mannopyranoside > pNp-β-galactopyranoside > pNp-β-xylopyranoside. pNp-β-mannopyranoside was the substrate exhibiting the lowest K m value [0.254 mM] with a K cat K m ratio comparable to that of pNp-β-glucopyranoside. This substrate specificity was distinct from previously reported β-glycosidases. We observed that the region in PK-Gly corresponding to the fifth α-helix and β-strand region of β-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to β-glycosidases and the C-terminal region similar to β-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.

Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized. Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M). Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate. Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.

In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase

Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (α 2β 2). The genes iorA and iorB which encode respective α and β subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions. IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay. The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment. IOR activity was detected neither from the extract of α subunit nor β subunit. The α and β subunits were mixed and then IOR activity was examined. Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced. The mixture of individually heat treated α and β subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR α and β subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass. Upon heat treatment, inactive α and β were converted to an active high molecular weight complex (195 kDa) which corresponds to the α 2β 2 structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the hetero-oligomerization of IOR subunits.

Archaeon Pyrococcus kodakaraensis KOD1: application and evolution

Archaea is the third domain which is phylogenetically differentiated from the other two domains, bacteria and eucarya. Hyperthermophile within the archaea domain has evolved most slowly retaining many ancestral features of higher eukaryotes. Pyrococcus kodakaraensis KOD1, which grows at 95 degrees C optimally, is a newly isolated hyperthermophilc archaeon. The KOD1 strain possesses a circular genome, whose size is estimated to be approximately 2,036 kb. KOD1 enzymes involved in the genetic information processing system, such as DNA polymerase, Rec protein, aspartyl tRNA synthetase and molecular chaperonin, share features of eukaryotic enzymes. Rapid and accurate PCR method by KOD1 DNA polymerase and enzyme stabilization system by KOD1 chaperonin are also introduced in this article.