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PubMed2006

Solution structure of a GAAG tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus.

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U.G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif the conformational stabilities are significantly different to each other It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.

Base Sequence↗

PubMed2000

Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production.

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6. 5 to 7.8 and at a temperature of over 95 degrees C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Alanine↗

PubMed2005

Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants.

Prolidases are dipeptidases specific for cleavage of Xaa-Pro dipeptides. Pyrococcus furiosus prolidase is a homodimer having one Co-bound dinuclear metal cluster per monomer with one tightly bound Co(II) site and the other loosely bound (Kd 0.24 mM). To identify which Co site is tight-binding and which is loose-binding, site-directed mutagenesis was used to modify amino acid residues that participate in binding the Co1 (E-313 and H-284), the Co2 site (D-209) or the bidentate ligand (E-327). Metal-content, enzyme activity and CD-spectra analyses of D209A-, H284L-, and E327L-prolidase mutants show that Co1 is the tight-binding and Co2 the loose-binding metal center.

Archaeal Proteins↗

PubMed2005

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study.

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.

Bacterial Proteins↗

PubMed2003

Purification and characterization of 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus: substrate specificity and primary structure analysis.

5'-Methylthioadenosine phosphorylase (MTAP) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The protein is a homoexamer of 180 kDa. The enzyme is highly thermoactive, with an optimum temperature of 125 degrees C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 degrees C and showing a half-life of 43 min at 130 degrees C. In the presence of 100 mM phosphate, the apparent T(m) (137 degrees C) increases to 139 degrees C. The enzyme is extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature. Thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present, since incubation with 0.8 M dithiothreitol significantly reduces the thermostability of the enzyme. N-Terminal sequence analysis of the purified enzyme is 100% identical to the predicted amino acid sequence of the gene PF0016 from the partially sequenced P. furiosus genome. The deduced amino acid sequence of the gene revealed a high degree of identity (52%) with human MTAP. Nevertheless, unlike human MTAP, MTAP from P. furiosus is not specific for 5'-methylthioadenosine, since it phosphorolytically cleaves adenosine, inosine, and guanosine. The calculated k(cat)/ K(m) values for 5'-methylthioadenosine and adenosine, about 20-fold higher than for inosine and guanosine, indicate that 6-amino purine nucleosides are preferred substrates of MTAP from P. furiosus. The structural features and the substrate specificity of MTAP from P. furiosus document that it represents a 5'-methylthioadenosine-metabolizing enzyme different from those previously characterized among Archaea, Bacteria, and Eukarya. The functional and structural relationships among MTAP from P. furiosus, human MTAP, and two putative MTAPs from P. furiosus and Sulfolobus solfataricus are discussed here for the first time.

Amino Acid Sequence↗

PubMed2005

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus.

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.

Amino Acid Sequence↗

PubMed2005

Calcium-induced tertiary structure modifications of endo-beta-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride.

The family 16 endo-beta-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-beta-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.

Amino Acid Sequence↗

PubMed1999

Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus.

The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli. The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps. The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours). pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe). It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups. Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product. The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions. Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast. The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule. These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group. In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule.

Cloning, Molecular↗

PubMed1990

Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.

Archaea↗

PubMed1995

Molecular characterization of the genes encoding the tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus and formaldehyde ferredoxin oxidoreductase from Thermococcus litoralis.

The hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis contain the tungstoenzymes aldehyde ferredoxin oxidoreductase, a homodimer, and formaldehyde ferredoxin oxidoreductase, a homotetramer. herein we report the cloning and sequencing of the P. furiosus gene aor (605 residues; M(r), 66,630) and the T. litoralis gene for (621 residues; M(r), 68,941).

Aldehyde Oxidoreductases↗

PubMed1996

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.

A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.

Amino Acid Sequence↗

PubMed2006

Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus.

Prolyl oligopeptidase (POP), the paradigm of a serine peptidase family, hydrolyses peptides, but not proteins. The thermophilic POP from Pyrococcus furiosus (Pfu) appeared to be an exception, since it hydrolysed large proteins. Here we demonstrate that the Pfu POP does not display appreciable activity against azocasein. The autolysis observed earlier was an artefact. We have also found that the pH-rate profile is different from that of the mammalian enzyme and the low pK(a) extracted from the curve represents the ionization of the catalytic histidine. We conclude that some oligopeptidases may be true endopeptidases, cleaving at disordered segments of proteins, but with very low efficacy.

Base Sequence↗

PubMed1999

A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.

The properties of the [4Fe-4S]2+/+ cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 A from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S]2+/+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe-4S]+ cluster in the wild-type protein, 80% S = 3/2 (E/D = 0.22, D = +3.3 cm(-1)) and 20% S = 1/2 (g = 2.10, 1.87, 1.80), is converted into a homogeneous S = 3/2 (E/D = 0.30, D = -0.7 cm(-1)) form in the A33Y variant. As the first example of a pure S = 3/2 [4Fe-4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S]+ clusters.

Circular Dichroism↗

PubMed2005

Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins.

The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95 degrees C, and at the lower end of the temperature range for significant growth, 72 degrees C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72 degrees C. This resulted in a 5-h lag phase, during which time little growth occurred. Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the P. furiosus genome showed that cells undergo three very different responses at 72 degrees C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72 degrees C). Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response. These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins. Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins. Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR. Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins. While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB. These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria. The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea.

Adaptation, Physiological↗

PubMed2004

Adsorption of an endoglucanase from the hyperthermophilic Pyrococcus furiosus on hydrophobic (polystyrene) and hydrophilic (silica) surfaces increases protein heat stability.

The interaction of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus with two types of surfaces, that is, hydrophobic polystyrene and hydrophilic silica, was investigated, and the adsorption isotherms were determined. The adsorbed hyperthermostable enzyme did not undergo loss of biological activity. A model was proposed for the mechanism of interaction of the enzyme with the surface based on the shape of the adsorption isotherm, the morphological characteristics of the enzyme, and the thermodynamic parameters of the system. The enzyme was irreversibly immobilized at the solid/liquid interface even at high temperatures, and most interestingly, it acquired further heat stabilization upon adsorption. The denaturation temperature increased from 108 degrees C in solution to 116 degrees C upon adsorption on hydrophilic silica particles. Adsorption on the hydrophobic polystyrene surface even shifted the denaturation temperature to 135 degrees C, the most extreme experimentally determined protein denaturation temperature ever reported. Maintenance of the biological function particularly at high temperatures is important for the development of solid substrate immobilized enzymes for applications in biocatalysis and biotechnology. This also presents an additional stabilization mechanism employed by nature where the extracellular hyperthermostable enzyme remains folded and active at the extreme temperatures of its natural environment by adsorption on the surface of rocks and other materials appearing in the surroundings of the microorganism.

Adsorption↗

PubMed2005

Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 A resolution. The crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 A.

Chitin↗

PubMed2006

Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of a catalytic domain of chitinase (PF1233 gene) from the hyperthermophilic archaeon Pyrococcus furiosus is reported. The recombinant protein, prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at the undulator beamline BL44XU at SPring-8 to a resolution of 1.50 angstroms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 90.0, b = 92.8, c = 107.2 angstroms.

Amino Acid Sequence↗

PubMed2004

Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules.

The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 A, and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining D2O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and Rfree = 21.7%. Several orientations of the O-D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N-H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the ND3+ terminus that may contribute to the unusual thermostability of this molecule.

Deuterium Exchange Measurement↗