SEARCH · PubMed Health

Results for “Pyrococcus horikoshii”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 163 records · Page 9Linked to original sources

PubMed2006

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C.

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T(d)) of nearly 150 degrees C, which exceeds the highest record determined by DSC by about 30 degrees C. To elucidate the mechanism of the ultra-high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 degrees C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T(d), up to 150 degrees C, for PhCutA1.

Archaeal Proteins↗

PubMed2003

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli.

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.

Acylation↗

PubMed2004

Crystal structure of a dodecameric tetrahedral-shaped aminopeptidase.

Protein turnover is an essential process in living cells. The degradation of cytosolic polypeptides is mainly carried out by the proteasome, resulting in 7-9-amino acid long peptides. Further degradation is usually carried out by energy-independent proteases like the tricorn protease from Thermoplasma acidophilum. Recently, a novel tetrahedral-shaped dodecameric 480-kDa aminopeptidase complex (TET) has been described in Haloarcula marismortui that differs from the known ring- or barrel-shaped self-compartmentalizing proteases. This complex is capable of degrading most peptides down to amino acids. We present here the crystal structure of the tetrahedral aminopeptidase homolog FrvX from Pyrococcus horikoshii. The monomer has a typical clan MH fold, as found for example in Aeromonas proteolytica aminopeptidase, containing a dinuclear zinc active center. The quaternary structure is built by dimers with a length of 100 A that form the edges of the tetrahedron. All 12 active sites are located on the inside of the tetrahedron. Substrate access is granted by pores with a maximal diameter of 10 A, allowing only small peptides and unfolded proteins access to the active site.

Amino Acid Sequence↗

PubMed2006

Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation.

Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 degrees C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 degrees C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.

Aeropyrum↗

PubMed2005

Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition.

Leucyl-tRNA synthetase (LeuRS) specifically recognizes the characteristic long variable arm and the discriminator base, A73, of tRNA(Leu) in archaea and eukarya. The LeuRS 'editing domain' hydrolyzes misformed noncognate aminoacyl-tRNA. Here we report the crystal structure of the archaeal Pyrococcus horikoshii LeuRS-tRNA(Leu) complex. The protruding C-terminal domain of LeuRS specifically recognizes the bases at the tip of the long variable arm. The editing domain swings from its tRNA-free position to avoid clashing with the tRNA. Consequently the tRNA CCA end can bend and reach the aminoacylation active site. The tRNA 3' region assumes two distinct conformations that allow A73 to be specifically recognized in different ways. One conformation is the canonical 'aminoacylation state.' The other conformation seems to be the 'intermediate state,' where the misaminoacylated 3' end has partially relocated to the editing domain.

Amino Acid Sequence↗

PubMed2005

Crystal structure of archaeal toxin-antitoxin RelE-RelB complex with implications for toxin activity and antitoxin effects.

The Escherichia coli chromosome encodes toxin-antitoxin pairs. The toxin RelE cleaves mRNA positioned at the A-site in ribosomes, whereas the antitoxin RelB relieves the effect of RelE. The hyperthermophilic archaeon Pyrococcus horikoshii OT3 has the archaeal homologs aRelE and aRelB. Here we report the crystal structure of aRelE in complex with aRelB determined at a resolution of 2.3 A. aRelE folds into an alpha/beta structure, whereas aRelB lacks a distinct hydrophobic core and extensively wraps around the molecular surface of aRelE. Neither component shows structural homology to known ribonucleases or their inhibitors. Site-directed mutagenesis suggests that Arg85, in the C-terminal region, is strongly involved in the functional activity of aRelE, whereas Arg40, Leu48, Arg58 and Arg65 play a modest role in the toxin's activity.

Antitoxins↗

PubMed2007

Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart.

Prefoldin (PFD) is a molecular chaperone that stabilizes and then delivers unfolded proteins to a chaperonin for facilitated folding. The PFD hexamer has undergone an evolutionary change in subunit composition, from two PFDalpha and four PFDbeta subunits in archaea to six different subunits (two alpha-like and four beta-like subunits) in eukaryotes. Here, we show by electron microscopy that PFD from the archaeum Pyrococcus horikoshii (PhPFD) selectively uses an increasing number of subunits to interact with nonnative protein substrates of larger sizes. PhPFD stabilizes unfolded proteins by interacting with the distal regions of the chaperone tentacles, a mechanism different from that of eukaryotic PFD, which encapsulates its substrate inside the cavity. This suggests that although the fundamental functions of archaeal and eukaryal PFD are conserved, their mechanism of substrate interaction have diverged, potentially reflecting a narrower range of substrates stabilized by the eukaryotic PFD.

Archaeal Proteins↗

PubMed2005

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase.

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.

Alcohol Oxidoreductases↗

PubMed2006

Common evolutionary origin of swapped-hairpin and double-psi beta barrels.

The core of swapped-hairpin and double-psi beta barrels is formed by duplication of a conserved betaalphabeta element, suggesting a common evolutionary origin. The path connecting the two folds is unclear as the two barrels are not interconvertible by a simple topological modification, such as circular permutation. We have identified a protein family whose sequence properties are intermediate to the two folds. The structure of one of these proteins, Pyrococcus horikoshii PhS018, is also built by duplication of the conserved betaalphabeta element but shows yet a third topology, which we name the RIFT barrel. This topology is widespread in the structure database and spans three folds of the SCOP classification, including the middle domain of EF-Tu and the N domain of F1-ATPase. We propose that swapped-hairpin beta barrels arose from an ancestral RIFT barrel by strand invasion and double-psi beta barrels by a strand swap. We group the three barrel types into a metafold, the cradle-loop barrels.

Amino Acid Sequence↗

PubMed2005

Molecular basis of alanine discrimination in editing site.

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).

Acylation↗

PubMed2005

Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.

Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.

Aminopeptidases↗

PubMed2006

Dimeric core structure of modular stator subunit E of archaeal H+ -ATPase.

Archaeal H(+)-ATPase (A-ATPase) is composed of an A(1) region that hydrolyzes ATP and an integral membrane part A(0) that conducts protons. Subunit E is a component of peripheral stator(s) that physically links A(1) and A(0) parts of the A-ATPase. Here we report the first crystal structure of subunit E of A-ATPase from Pyrococcus horikoshii OT3 at 1.85 A resolution. The protomer structure of subunit E represents a novel fold. The quaternary structure of subunit E is a homodimer, which may constitute the core part of the stator. To investigate the relationship with other stator subunit H, the complex of subunits EH was prepared and characterized using electrophoresis, mass spectrometry, N-terminal sequencing and circular dichroism spectroscopy, which revealed the polymeric and highly helical nature of the EH complex with equimolar stoichiometry of both the subunits. On the basis of the modular architecture of stator subunits, it is suggested that both cytoplasm and membrane sides of the EH complex may interact with other subunits to link A(1) and A(0) parts.

Circular Dichroism↗

PubMed2005

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method.

A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr Kalpha radiation (2.29 A). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr Kalpha radiation, the anomalous signal |DeltaF|/|F| of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique.

Absorption↗

PubMed2005

Crystal structure of the NAD biosynthetic enzyme quinolinate synthase.

A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction.

Archaeal Proteins↗

PubMed2006

Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase.

To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA(Phe), indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3'-terminal adenosine, reduced Tyr-tRNA(Phe) deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

Amino Acid Sequence↗

PubMed2005

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

Base Sequence↗

PubMed2004

An archaeal histone-like protein as an efficient DNA carrier in gene transfer.

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.

Animals↗

PubMed2006

The first crystal structure of L-threonine dehydrogenase.

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.

Alcohol Oxidoreductases↗