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At least 145 records · Page 8Linked to original sources

PubMed2002

Crystallization and preliminary X-ray diffraction analysis of L-aminoacylase from the hyperthermophilic archaeon Thermococcus litoralis.

The enzyme L-aminoacylase catalyses the hydrolysis of N-acyl-L-amino acids from peptides or proteins. The recombinant enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been purified to homogeneity. This zinc-containing enzyme has been crystallized from ammonium sulfate using the sitting-drop vapour-diffusion method. The crystals diffract to 2.8 A resolution and belong to the rhombohedral space group R32, with unit-cell parameters a = b = 102.4, c = 178.5 A, gamma = 120 degrees in a hexagonal lattice setting. The asymmetric unit contains one enzyme monomer, containing a single zinc ion. Two synchrotron data sets have been collected at a remote wavelength and at the maximum f'wavelength for zinc. This has allowed the position of the metal to be identified in anomalous Patterson maps.

Amidohydrolases↗

PubMed2002

Crystallization and preliminary X-ray characterization of archaeal group II chaperonin alpha-subunit from Thermococcus strain KS-1.

The archaeal group II chaperonin from Thermococcus strain KS-1 is composed of two kinds of subunits (alpha and beta). Each of the recombinant subunits was individually expressed in Escherichia coli and purified as homo-hexadecamers of each subunit. Both homo-oligomers facilitate the refolding of denatured proteins in vitro in an ATP-dependent manner. A mutant alpha-subunit homo-oligomer with two amino-acid substitutions, which has the ability to capture the unfolded protein but lacks the ability to refold the unfolded protein, was crystallized in two different conditions. One crystal form was obtained from a high-concentration solution of ammonium sulfate and grew to maximum dimensions of 0.15 x 0.15 x 0.4 mm. The crystals of this form belonged to the tetragonal space group P42(1)2, with unit-cell parameters a = b = 209.3, c = 156.1 A, and diffracted X-rays to 2.4 A resolution with synchrotron radiation. The other form was crystallized from a polyethylene glycol 6000 solution and belonged to the tetragonal space group, with unit-cell parameters a = b = 220.8, c = 182.4 A. This form only diffracts X-rays to 6 A resolution. Diffraction data collected from the former crystal enabled initial successful phases to be obtained by the molecular-replacement method.

Archaeal Proteins↗

PubMed2002

Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.

Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

Archaeal Proteins↗

PubMed2003

Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1.

The 26 kDa TBP (TATA-binding protein) interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-TIP26) is a possible transcriptional regulatory protein in Thermococcales. Here, the crystallization of both the native and selenomethionine-substituted proteins and data collection are described. The native crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 73.83, c = 86.41 A, and diffract to 2.2 A using synchrotron radiation. MAD data was collected and a Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms.

Archaeal Proteins↗

PubMed1998

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Crystallization↗

PubMed1999

Crystallization and preliminary X-ray diffraction studies of pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis.

Pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis has been crystallized in a form suitable for X-ray diffraction from ammonium sulfate or ammonium dihydrogen orthophosphate using the vapour-phase diffusion method. Crystals from both precipitants are of the orthorhombic space group P21212 with unit-cell dimensions a = 94.06, b = 149.06, c = 73.54 A. A complete data set to 2.8 A resolution has been collected from crystals grown from ammonium sulfate.

Crystallization↗

PubMed1999

Crystallization and preliminary x-ray diffraction studies of hyperthermostable glutamate dehydrogenase from Thermococcus profundus.

Recombinant glutamate dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus, was crystallized in the presence of both polyethylene glycol 8000 and lithium sulfate. Four types of crystals having different morphologies appeared in the crystallization trials; however, only one type was suitable for X-ray crystal structure analysis. The crystal belonged to the monoclinic space group P2(1) and the unit-cell parameters were a = 112.99, b = 163.70, c = 133.07 A, beta = 113.46 degrees at 110 K. The calculated V(M) value of 3.42 A(3) Da(-1) was acceptable when one hexamer of the enzyme, which was the physiological functional unit, occupied a crystallographic asymmetric unit. X-ray diffraction intensity data were collected to a resolution of 2.25 A with good statistics at the BL44B2 beamline of SPring-8.

Crystallization↗

PubMed2003

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer.

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.

Amino Acid Sequence↗

PubMed2001

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis.

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.

Enzyme Stability↗

PubMed2001

Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition.

To study the difference in expression of the chaperonin alpha- and beta-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The beta-subunit was significantly more abundant with increasing temperature (maximum at 93 degrees C), whereas the alpha-subunit was not. Native PAGE with Western blot analysis indicated that the natural chaperonins in the crude extracts of T. KS-1 cells grown between 65 degrees C and 95 degrees C migrate as single bands with different mobility. The recombinant alpha- and beta-subunit homo-oligomers migrated differently from each other and from natural chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero-oligomer. These results indicate that chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the beta-subunit may be adapted to a higher temperature than the alpha-subunit. T. KS-1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.

Blotting, Western↗

PubMed2006

Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden-Meyerhof pathway in Thermococcus kodakarensis.

We have carried out a genetic analysis on pyruvate kinase (PykTk) and phosphoenolpyruvate synthase (PpsTk) in the hyperthermophilic archaeon, Thermococcus kodakarensis. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the former utilizing ADP, while the latter is dependent on AMP and phosphate. Enzyme activities and transcript levels of both PykTk and PpsTk increased in T. kodakarensis under glycolytic conditions when compared with cells grown on pyruvate or amino acids. Using KW128, a tryptophan auxotrophic mutant with a trpE gene disruption, as a host strain, we obtained mutant strains with single gene disruptions in either the pykTk (Deltapyk strain) or ppsTk (Deltapps strain) gene. Specific growth rates and cell yields were examined in various media and compared with the host KW128 strain. The results indicated that both enzymes participated in pyruvate metabolism, but were not essential. In the presence of maltooligosaccharides, the Deltapyk strain displayed a 15% decrease in growth rate compared with the host strain, indicating that PykTk does participate in glycolysis. However an even more dramatic effect was observed in the Deltapps strain in that the strain could not grow at all on maltooligosaccharides. The results clearly indicate that, in contrast to the conventional EM pathway dependent on pyruvate kinase, PEP synthase is the essential enzyme for the glycolytic conversion of PEP to pyruvate in T. kodakarensis. The physiological roles of the two enzymes under various growth conditions are discussed.

Archaeal Proteins↗

PubMed2000

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans.

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.

Amino Acid Sequence↗

PubMed2006

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

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

Base Sequence↗

PubMed2006

Ca2+-dependent maturation of subtilisin from a hyperthermophilic archaeon, Thermococcus kodakaraensis: the propeptide is a potent inhibitor of the mature domain but is not required for its folding.

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80 degrees C. This maturation process was completed within 30 min at 80 degrees C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80 degrees C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80 degrees C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only approximately 5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 +/- 3.0 nM at 20 degrees C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.

Archaeal Proteins↗

PubMed1999

Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter. The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C. The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.

Amino Acid Sequence↗

PubMed2001

Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus.

The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.

2,6-Dichloroindophenol↗

PubMed2005

Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis.

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (DeltapyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (DeltapyrF DeltatrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (DeltapyrF DeltatrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (DeltapyrF DeltatrpE DeltahisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

Archaeal Proteins↗

PubMed2006

A novel branching enzyme of the GH-57 family in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the alpha-1,4 linkage and its subsequent transfer to the alpha-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain alpha-glucans, indicating that the reaction product contained many alpha-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70 degrees C, and thermostability up to 90 degrees C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436DeltaH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436DeltaH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

Bacterial Proteins↗