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PubMed2005

Comparison of small- and large-scale expression of selected Pyrococcus furiosus genes as an aid to high-throughput protein production.

As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.

Bacterial Proteins↗
PubMed2002

Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk.

Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C. The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.

Animals↗
PubMed2000

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus.

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.

Aminopeptidases↗
PubMed2002

Resonance Raman characterization of the mononuclear iron active-site vibrations and putative electron transport pathways in Pyrococcus furiosus superoxide reductase.

The resonance Raman spectrum of oxidized wild-type P. furiosus SOR at pH 7.5 and 10.5 has been investigated using excitation wavelengths between 406 and 676 nm, and vibrational modes have been assigned on the basis of isotope shifts resulting from global replacements of (32)S with (34)S, (14)N with (15)N, (56)Fe with (54)Fe, and exchange into a H(2)(18)O buffer. The results are interpreted in terms of the crystallographically defined active-site structure involving a six-coordinate mononuclear Fe center with four equatorial histidine ligands and axial cysteine and monodentate glutamate ligands (Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. C. (2000) Biochemistry 39, 2499-2508). Excitation into the intense (Cys)S(p(pi))-to-Fe(d(pi)) CT transition centered at 660 nm results in strong enhancement of modes at 298 cm(-1) and 323 cm(-1) that are assigned to extensively mixed cysteine S-C(beta)-C(alpha) bending and Fe-S(Cys) stretching modes, respectively. All other higher-energy vibrational modes are readily assigned to overtone or combination bands or to fundamentals corresponding to internal modes of the ligated cysteine. Weak enhancement of Fe-N(His) stretching modes is observed in the 200-250 cm(-1) region. The enhancement of internal cysteine modes and Fe-N(His) stretching modes are a consequence of a near-planar Fe-S-C(beta)-C(alpha)-N unit for the coordinated cysteine and significant (His)N(p(pi))-Fe(d(xy))-(Cys)S(p(pi)) orbital overlap, respectively, and have close parallels to type 1 copper proteins. By analogy with type 1 copper proteins, putative superexchange electron-transfer pathways to the mononuclear Fe active site are identified involving either the tyrosine and cysteine residues or the solvent-exposed deltaN histidine residue in a Y-C-X-X-H arrangement. Studies of wild-type at pH 10.5 and the E14A variant indicate that the resonance Raman spectrum is remarkably insensitive to changes in the ligand trans to cysteine and hence are inconclusive concerning the origin of the alkaline transition and the nature of sixth Fe ligand in the E14A variant.

Binding Sites↗
PubMed1993

The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence.

Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P. furiosus. Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P. furiosus gdh gene was identified, cloned into Escherichia coli and sequenced. The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site. Using antiserum raised against purified GDH, expression of gdh was observed in E. coli. The deduced primary sequence of the P. furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%).

Amino Acid Sequence↗
PubMed1998

Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Aminopeptidases↗
PubMed1990

The hyperthermophilic archaebacterium, Pyrococcus furiosus. Development of culturing protocols, perspectives on scaleup, and potential applications.

From this brief discussion, it is clear that there are many obstacles to overcome before hyperthermophilic archaebacteria will be an important aspect of biotechnology. Nevertheless, the prospects are intriguing. The nature of the environments that harbor these organisms and the consequent requirements for their controlled culture suggest that chemical and biochemical engineers can play an important role in elucidating their scientific and technological aspects.

Archaea↗
PubMed2001

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.

Amino Acid Sequence↗
PubMed2000

Carbamate kinase can replace in vivo carbamoyl phosphate synthetase. Implications for the evolution of carbamoyl phosphate biosynthesis.

The exclusive involvement of carbamate kinase (CK) in fermentative ATP production and of carbamoyl phosphate synthetase (CPS) in the production of carbamoyl phosphate (CP) for pyrimidines and arginine biosynthesis was challenged by the finding of CK as the only activity synthesising CP in the archaea Pyrococcus furiosus and Pyrococcus abyssi. We now show that CK can replace CPS in vivo: transformation of Escherichia coli devoid of the CPS gene with plasmids encoding the CK from P. furiosus or from Enterococcus faecalis (which uses CK for making ATP) restores the ability of CPS-deficient E. coli to grow in the absence of arginine and uracil if ammonia and bicarbonate are present.

Ammonia↗
PubMed2005

Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment.

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.

DNA Transposable Elements↗
PubMed1996

Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus.

A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed. In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C. In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T. The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant. From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera. The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli. The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues. This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments. It is noteworthy that an opposite trend, i.e. a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Animals↗
PubMed2003

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi.

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

Adaptation, Physiological↗
PubMed1996

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.

Amino Acid Sequence↗