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PubMed2001

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Amino Acid Sequence↗

PubMed2001

Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.

Adaptation, Physiological↗

PubMed1995

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A. Consideration of the values of V(m) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

Journal Article↗

PubMed1997

Repair of extensive ionizing-radiation DNA damage at 95 degrees C in the hyperthermophilic archaeon Pyrococcus furiosus.

We investigated the capacity of the hyperthermophile Pyrococcus furiosus for DNA repair by measuring survival at high levels of 60Co gamma-irradiation. The P. furiosus 2-Mb chromosome was fragmented into pieces ranging from 500 kb to shorter than 30 kb at a dose of 2,500 Gy and was fully restored upon incubation at 95 degrees C. We suggest that recombination repair could be an extremely active repair mechanism in P. furiosus and that it might be an important determinant of survival of hyperthermophiles at high temperatures.

Archaea↗

PubMed1994

Indolepyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. A new enzyme involved in peptide fermentation.

Pyrococcus furiosus is a strictly anaerobic archaeon that grows optimally at 100 degrees C by a fermentative-type metabolism in which complex peptide mixtures such as yeast extract and Tryptone, and also certain sugars, are oxidized to organic acids, H2 and CO2. Enzymes involved in the utilization of peptides such as proteases, aromatic amino transferases, and glutamate dehydrogenase have been previously purified from this organism. It is shown here that P. furiosus also contains significant cytoplasmic concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase (IOR). This catalyzes the oxidative decarboxylation of aryl pyruvates, which are generated by the transamination of aromatic amino acids, to the corresponding aryl acetyl-CoA. IOR is a tetramer (alpha 2 beta 2) of two identical subunits (66,000 and 23,000 Da) with a molecular weight of 180,000. The enzyme contains one molecule of thiamine pyrophosphate and four [4Fe-4S]2+,1+ and one [3Fe-4S]0,1+ cluster, as determined by iron analyses and EPR spectroscopy. Significant amounts of other metals such as copper and zinc were not detected. IOR was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.5-10.5 (at 80 degrees C). The enzyme was sensitive to inactivation by O2, losing 50% of its activity after exposure to air for 20 min at 23 degrees C, and was quite thermostable, with a half-life of activity at 80 degrees C (under anaerobic conditions) of about 80 min. The Km values (in microM) for indolepyruvate, p-hydroxyphenylpyruvate, phenylpyruvate, CoASH, and P. furiosus ferredoxin, the physiological electron carrier, were 250, 110, 90, 17, and 48, respectively. IOR was inhibited by KCN (apparent Ki = 7.5 mM), but not by CO (1 atm). An enzyme analogous to IOR has not been reported previously. Curiously, it has few properties in common with the pyruvate ferredoxin oxidoreductase of P. furiosus, even though the two enzymes catalyze virtually identical reactions. In fact, of known ketoacid oxidoreductases, the catalytic mechanism of IOR appears to be most similar to that of the pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium Thermotoga maritima.

Amino Acid Sequence↗

PubMed1997

The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,.

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.

Amino Acid Sequence↗

PubMed2003

Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus.

Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

Archaeal Proteins↗

PubMed2006

Efficient solubilization, purification of recombinant extracellular alpha-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli.

The gene encoding the Pyrococcus furiosus extracellular alpha-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant alpha-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90 degrees C treatment for 3 min in Britton-Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher than previously reported.

Enzyme Stability↗

PubMed2001

Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus.

A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain. Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales. There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins. Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 m guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cis-proline) had disappeared. Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.

Amino Acid Sequence↗

PubMed2001

Crystallization and characterization of the prolidase from Pyrococcus furiosus.

The prolidase (proline-specific amino dipeptidase) from the hyperthermophilic archaeon Pyrococcus furiosus has been crystallized. The enzyme has been shown to be a homodimer and to require two Co atoms per subunit for optimum activity. Two crystal forms have been obtained under similar growth conditions. Both are monoclinic, space group P2(1). Form I has unit-cell parameters a = 130.4, b = 97.4, c = 129.9 A, beta = 118.3 degrees. Form II has a smaller unit cell, with a = 56.5, b = 97.3, c = 70.0 A, beta = 97.1 degrees. If the crystal density is assumed to lie near the center of the normal range then the form I crystals will have four dimers per asymmetric unit, whereas the form II crystals will have only one dimer in each asymmetric unit. Diffraction data have been recorded from native form I and form II crystals to resolutions of 3.2 and 1.95 A, respectively.

Crystallization↗

PubMed2006

Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation.

The remarkable survival of the hyperthermophilic archaeon Pyrococcus furiosus to ionizing radiation was previously demonstrated. Using a time course study and whole-genome microarray analyses of mRNA transcript levels, the genes and regulatory pathways involved in the repair of lesions produced by ionizing irradiation (oxidative damage and DNA strand breaks) in P. furiosus were investigated. Data analyses showed that radA, encoding the archaeal homolog of the RecA/Rad51 recombinase, was moderately up regulated by irradiation and that a putative DNA-repair gene cluster was specifically induced by exposure to ionizing radiation. This novel repair system appears to be unique to thermophilic archaea and bacteria and is suspected to be involved in translesion synthesis. Genes that encode for a putative Dps-like iron-chelating protein and two membrane-bound oxidoreductases were differentially expressed following gamma irradiation, potentially in response to oxidative stress. Surprisingly, the many systems involved in oxygen detoxification and redox homeostasis appeared to be constitutively expressed. Finally, we identified several transcriptional regulators and protein kinases highly regulated in response to gamma irradiation.

Archaeal Proteins↗

PubMed2003

Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA.

Two mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site-specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant PfuPCNAs were determined. Although the wild-type PCNA forms a toroidal trimer with intermolecular hydrogen bonds between the N- and C-terminal domains, the mutant PfuPCNAs exist as V-shaped dimers through intermolecular hydrogen bonds between the two C-terminal domains in the crystal. Because the mutated residues are involved in the intermolecular ion pairs through their side chains in the wild-type PfuPCNA, these ion pairs seem to play a key role in maintaining the toroidal structure of the PfuPCNA trimer. The comparison of the crystal structures revealed intriguing conformational flexibility of each domain in the PfuPCNA subunit. This structural versatility of PCNA may be involved in the mechanisms for ring opening and closing.

Amino Acid Substitution↗

PubMed2005

Salvaging Pyrococcus furiosus protein targets at SECSG.

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.

Archaeal Proteins↗

PubMed2002

Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus.

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(II). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest K(m) values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (k(cat)/K(m)) of PfMetAP-II for MP-p-NA at 30 degrees C was 799 500 and 340 930 M(-1) s(-1) for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 50 +/- 15 and 20 +/- 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 +/- 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be approximately 85 degrees C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 degrees C are as follows: DeltaG++ = 46.23 kJ/mol, DeltaH++ = 10.79 kJ/mol, and DeltaS++ = -119.72 J.mol(-1).K(-1) for Co(II)-loaded PfMetAP; DeltaG++ = 46.44 kJ/mol, DeltaH++ = 16.94 kJ/mol, and DeltaS++ = -99.67 J.mol(-1).K(-1) for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (> 50 degrees C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degrees C) than Co(II)-loaded PfMetAP-II.

Aminopeptidases↗

PubMed2004

The 1.5 A resolution crystal structure of [Fe3S4]-ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

The structure of [Fe(3)S(4)]-ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus has been determined to 1.5 A resolution from a crystal belonging to space group P2(1) with two molecules in the asymmetric unit. The structure has been solved with molecular replacement by use of the ferredoxin from Thermotoga maritima. The fold is similar to that of related monocluster ferredoxins and contains two double-stranded antiparallel beta-sheets and two alpha-helices. The hydrophobic interaction between Trp2 and Tyr46 is confirmed, linking the C-terminus to the longer alpha-helix. The structure contains a double-conformation disulfide bond existing in a left-handed and a right-handed spiral conformation. The crystal packing reveals a beta-sheet interaction, which supports the suggestion that P. furiosus ferredoxin is a functional dimer. The extraordinary thermostability of P. furiosus ferredoxin is further discussed.

Amino Acid Sequence↗

PubMed2001

Anaerobic desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus--a new method for rubber recycling.

The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber.

Biotechnology↗

PubMed1999

On the prosthetic groups of the NiFe sulfhydrogenase from Pyrococcus furiosus: topology, structure, and temperature-dependent redox chemistry.

The sulfhydrogenase complex of Pyrococcus furiosus is an alpha beta gamma delta heterotetramer with both hydrogenase activity (borne by the alpha delta subunits) and sulfur reductase activity (carried by the beta gamma subunits). The beta-subunit contains at least two [4Fe-4S] cubanes and the gamma-subunit contains one [2Fe-2S] cluster and one FAD molecule. The delta-subunit contains three [4Fe-4S] cubanes and the alpha-subunit carries the NiFe dinuclear center. Only three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other clusters presumably have reduction potentials well below that of the H+/H2 couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent reduction potentials.

Amino Acid Sequence↗

PubMed2006

Entrapment in E. coli improves the operational stability of recombinant beta-glycosidase CelB from Pyrococcus furiosus and facilitates biocatalyst recovery.

beta-Glycosidase CelB from the hyperthermophilic archaeon Pyrococcus furiosus is a versatile biocatalyst that has been used for the hydrolysis and synthesis of beta-d-glycosidic compounds at high temperatures and in non-conventional solvents. In spite of its outstanding thermal stability, CelB is prone to inactivation in the presence of reducing sugars and through recirculation in loop enzyme reactors. Entrapment into E. coli cells was used here to improve the stability of recombinant CelB under conditions promoting strong inactivation. Glutardialdehyde-mediated protein cross-linking or rigidification of the cell membrane by adding magnesium ions was required to prevent release of CelB from within the cell into the bulk solution. In the presence of 1M glucose or when applying recirculation rates of 2.6 min(-1), the entrapped enzyme was around two-fold more stable at 80 degrees C than free CelB. The significance of the stabilisation was attenuated by the decrease in CelB initial activity which was due to cross-linking and glutardialdehyde concentration-dependent. Entrapment facilitated downstream processing of CelB and biocatalyst recovery in repeated batchwise conversions of lactose at elevated temperatures.

Bacterial Proteins↗