Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pyrococcus horikoshii”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2003

Alkyl hydroperoxide reductase dependent on thioredoxin-like protein from Pyrococcus horikoshii.

Pyrococcus horikoshii is an obligate anaerobic hyperthermophilic archaeon. In P. horikoshii cells, a hydroperoxide reductase homologue ORF (PH1217) was found to be induced by oxygen. The recombinant protein, which was expressed in E. coli under aerobic conditions, exhibited no activity. However, the recombinant protein prepared under semi-anaerobic conditions exhibited alkyl hydroperoxide reductase activity. Furthermore, it was clarified that it was coupled with the thioredoxin-like system in P. horikoshii. Western blot analysis revealed that the protein was induced by oxygen and hydrogen peroxide. This protein seems to be sensitive to oxygen but forms a thioredoxin-dependent system to eliminate reactive oxygen species in P. horikoshii.

Amino Acid Sequence↗
PubMed2005

Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.

Pyrococcus horikoshii open reading frame PH1527 encodes a 39014 Da protein that shares about 30% identity with endoglucanases and members of the M42 peptidase family. Analytical ultracentrifugation and electron microscopy studies showed that the purified recombinant protein forms stable, large dodecameric complexes with a tetrahedral shape similar to the one described for DAP, a deblocking aminopeptidase that was characterized in the same organism. The two related proteins were named PhTET1 (for DAP) and PhTET2 (for PH1527). The substrate specificity and the mode of action of the PhTET2 complex were studied in detail and compared to those of PhTET1 and other assigned M42 peptidases. When assayed with short chromogenic peptides, PhTET2 was found to be an aminopeptidase, with a clear preference for leucine as the N-terminal amino acid. However, the enzyme can cleave moderately long polypeptide substrates of various compositions in a fairly unspecific manner. The hydrolytic mechanism was found to be nonprocessive. The enzyme has neither carboxypeptidase nor endoproteolytic activities, and it is devoid of N-terminal deblocking activity. PhTET2 was inhibited in the presence of EDTA and bestatin, and cobalt was found to be an activating metal. The PhTET2 protein is a highly thermostable enzyme that displays optimal activity around 100 degrees C over a broad pH array.

Amino Acid Sequence↗
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↗
PubMed2003

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii.

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.

Amino Acid Sequence↗
PubMed2001

Crystallization and preliminary X-ray analysis of aspartate racemase from Pyrococcus horikoshii OT3.

Aspartate racemase from Pyrococcus horikoshii OT3 (P. AspR) has been crystallized in three crystal forms by the sitting-drop vapour-diffusion method. The crystals belong to the space groups P2(1), P2(1)2(1)2(1) and P3(1)21 (or P3(2)21). The crystals of space group P2(1) diffract X-rays beyond 1.7 A resolution under 90 K liquid-nitrogen cryoconditions with synchrotron radiation and were selected for structure determination. Two heavy-atom derivatives of this crystal form were obtained by the soaking method, which afforded the initial electron-density map.

Amino Acid Isomerases↗
PubMed2004

Oxidative stress response in an anaerobic hyperthermophilic archaeon: presence of a functional peroxiredoxin in Pyrococcus horikoshii.

Oxidative stress response in an anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was analyzed by two-dimensional gel electrophoresis. When P. horikoshii was grown on medium supplemented with air, a marked increase in the level of a 25-kDa protein was observed in comparison with cells grown under anaerobic conditions. The N-terminal amino acid sequence of the protein was determined to be VVIGEKFPEVEVKTTHGVIKLPDYF, which coincides with that of the putative alkyl hydroperoxide reductase that has been predicted in the genome database of P. horikoshii. The gene (PH1217) encoding the protein was cloned and expressed in Escherichia coli. The produced enzyme was a hyperthermostable peroxiredoxin whose activity was not lost after incubation at 90 degrees C for 20 min. The enzyme catalyzes the reduction of cumene hydroperoxide and hydrogen peroxide using dithiothreitol as an electron donor. Northern blot analysis revealed that the transcription of the gene increased by the addition of exogenous oxygen and by the addition of an oxidative stress-inducing reagent, and reached maximum within 30 min. These results suggest that the peroxiredoxin plays an important role in the peroxide-scavenging system in an anaerobic archaeon P. horikoshii.

Amino Acid Sequence↗
PubMed2004

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.

Amino Acid Sequence↗
PubMed2000

Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution.

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3.

The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45 degrees C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90 degrees C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD.

Adenosine Triphosphatases↗
PubMed2004

Crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 71.7, b = 86.5, c = 92.5 A, alpha = beta = gamma = 90 degrees. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A resolution in-house.

Crystallization↗
PubMed2004

Crystallization and preliminary X-ray crystallographic study of leucyl-tRNA synthetase from the archaeon Pyrococcus horikoshii.

The leucyl-tRNA synthetase (LeuRS) from the archaeon Pyrococcus horikoshii was overexpressed in a C-terminally truncated form in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals belong to the rhombohedral space group R3, with unit-cell parameters a = b = 186.20, c = 91.43 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit contains one molecule of LeuRS, with a corresponding crystal volume per protein weight of 3.2 A3 Da(-1) and a solvent content of 60.7%. A data set diffracting to 2.2 A resolution was collected from a single crystal at 100 K. Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the SAD phasing method.

Crystallography, X-Ray↗
PubMed2005

Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii.

In the genome data base of the hyperthermophilic archaeon Pyrococcus horikoshii, an open reading frame with sequence homology to a gene encoding alcohol dehydrogenase was found. It was demonstrated that the encoded enzyme was a thermostable L-threonine dehydrogenase which can oxidize the hydroxy alkyl residue of L-threonine associated with the reduction of NAD+ or NADP+. This enzyme is a member of the zinc-containing L-threonine dehydrogenase family. One enzyme molecule contained one zinc atom, and this metal was considered to contribute to the hyperthermostablility of the enzyme. The reaction of the enzyme proceeded via a sequential mechanism. The Michaelis constants (Km) for L-threonine and NAD+ were 0.013 and 0.010 mM, respectively, and the maximum reaction rate (Vmax) was 1.75 mmol NADH formed/min/mg-protein at 65 degrees C. The Km values for both L-threonine and NADP+ were larger than those for L-threonine and NAD+ with a similar Vmax value. These results indicate that the enzyme has lower affinity to NADP+ than to NAD+, and the binding affinity for L-threonine depends on the coenzymes.

Alcohol Oxidoreductases↗
PubMed2004

Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3.

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.

Amino Acid Sequence↗
PubMed2002

Hyperthermostable endoglucanase from Pyrococcus horikoshii.

An endoglucanase homolog from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, and its enzymatic characteristics were examined. The expressed protein was a hyperthermostable endoglucanase which hydrolyzes celluloses, including Avicel and carboxymethyl cellulose, as well as beta-glucose oligomers. This enzyme is the first endoglucanase belonging to glycosidase family 5 found from Pyrococcus species and is also the first hyperthermostable endoglucanase to which celluloses are the best substrates. This enzyme is expected to be useful for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products.

Amino Acid Sequence↗
PubMed2005

New deblocking aminopeptidases from Pyrococcus horikoshii.

It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 degrees C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.

Amino Acid Sequence↗
PubMed2002

Crystallization and preliminary X-ray analysis of recombinant histone HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Recombinant archaeal histone from the hyperthermophile Pyrococcus horikoshii OT3 (HPhA) was crystallized by the hanging-drop vapour-diffusion method. Crystals grew at 291 K in 200 mM (NH(4))(2)SO(4), 100 mM sodium acetate buffer pH 4.6, 19% PEG 4000. Diffraction data were obtained to a resolution of 2.3 A from a single frozen crystal, which belonged to space group P2(1) with unit-cell parameters a = 34.99, b = 46.89, c = 35.02 A, alpha = gamma = 90, beta = 104 degrees. The asymmetric unit contained two molecules and had a solvent content of approximately 35%.

Crystallization↗
PubMed2002

Active site of deblocking aminopeptidase from Pyrococcus horikoshii.

New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion.

Amino Acid Sequence↗
PubMed2000

Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.

Amino Acid Sequence↗