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F T Robb

Publications and source records attributed to F T Robb.

At least 37 records · Page 2Linked to original sources
PubMed1999

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Adaptation of proteins from hyperthermophiles to high pressure and high temperature.

Further clarification of the adaptations permitting the persistence of life at temperatures above 100 degrees C depends in part on the analysis of adaptive mechanisms at the protein level. The hyperthermophiles include both Bacteria and Archaea, although the majority of isolates growing at or above 100 degrees C are Archaea. Newly described adaptive features of hyperthermophiles include proteins whose structural integrity persists at temperatures up to 200 degrees C, and under elevated hydrostatic pressure, which in some cases adds significant increments of stability.

Adaptation, Physiological↗
PubMed1998

Protein thermostability above 100 degreesC: a key role for ionic interactions.

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Amino Acid Sequence↗
PubMed1998

Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family.

The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.

Amino Acid Sequence↗
PubMed1998

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough.

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.

Japan↗
PubMed1997

Ribonucleotide reductase in the archaeon Pyrococcus furiosus: a critical enzyme in the evolution of DNA genomes?

Ribonucleotide reductase (RNR), the enzyme responsible for deoxyribonucleotide synthesis, has been isolated from Pyrococcus furiosus, a deeply branching hyperthermophilic, strictly anaerobic archaeon. Its gene has been cloned, sequenced, and shown to harbor two insertions encoding inteins. The purified enzyme absolutely requires adenosylcobalamin for activity, a trait that defines it as a member of class II (adenosyl-cobalamin-dependent) prokaryotic RNRs. On the other hand, the archaeal RNR has significant amino acid sequence homology with class I (aerobic non-heme iron-dependent) and class III (anaerobic iron-sulfur-dependent) RNRs present in eukaryotes and bacteria, respectively. It is proposed that this enzyme may be the closest possible relative of the original RNR, which allowed the key "RNA world" to "DNA world" transition, and that the different classes of present-day RNRs are the products of divergent evolution.

Amino Acid Sequence↗
PubMed1997

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases.

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types-and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166-170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria.

Amino Acid Sequence↗
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↗
PubMed1997

Characterization of the reverse gyrase from the hyperthermophilic archaeon Pyrococcus furiosus.

The reverse gyrase gene rgy from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and sequenced. The gene is 3,642 bp (1,214 amino acids) in length. The deduced amino acid sequence has relatively high similarity to the sequences of the Methanococcus jannaschii reverse gyrase (48% overall identity), the Sulfolobus acidocaldarius reverse gyrase (41% identity), and the Methanopynrus kandleri reverse gyrase (37% identity). The P. furiosus reverse gyrase is a monomeric protein, containing a helicase-like module and a type I topoisomerase module, which resembles the enzyme from S. acidocaldarius more than that from M. kandleri, a heterodimeric protein encoded by two separate genes. The control region of the P. furiosus rgy gene contains a typical archaeal putative box A promoter element which is located at position -26 from the transcription start identified by primer extension experiments. The initiating ATG codon is preceded by a possible prokaryote-type ribosome-binding site. Purified P. furiosus reverse gyrase has a sedimentation coefficient of 6S, suggesting a monomeric structure for the native protein. The enzyme is a single polypeptide with an apparent molecular mass of 120 kDa, in agreement with the gene structure. The sequence of the N terminus of the protein corresponded to the deduced amino acid sequence. Phylogenetic analysis indicates that all known reverse gyrase topoisomerase modules form a subgroup inside subfamily IA of type I DNA topoisomerases (sensu Wang [J. C. Wang, Annu. Rev. Biochem. 65:635-692, 1996]). Our results suggest that the fusion between the topoisomerase and helicase modules of reverse gyrase occurred before the divergence of the two archaeal phyla, Crenoarchaeota and Euryarchaeota.

Amino Acid Sequence↗
PubMed1996

Crystallization of the glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

The NADP(+)-dependent glutamate dehydrogenase from Thermococcus litoralis has been crystallized by the hanging-drop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group C2 with unit-cell dimensions a = 142.7, b = 202.0, c = 125.8 A with beta = 113.1 degrees with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a half-life of 2 h at 373 K and a comparison of this structure with that of other GluDH's from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.

Journal Article↗
PubMed1995

Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis.

In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.

Amino Acid Sequence↗
PubMed1995

A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous to members of the prolyl oligopeptidase family of proteases.

The mlr-2 gene from the hyperthermophilic archaeum Pyrococcus furiosus was identified from a family of clones whose expression was influenced by the presence of maltose in the medium. The sequence of 2100 bp of DNA containing mlr-2 and its flanking regions revealed a 616-amino-acid (71 kDa) open reading frame (ORF). The ORF's initiation codon appeared 10 nt into the mlr-2 message and was not preceded by any apparent ribosome-binding site. The deduced product shared homology with prolyl endopeptidases from both eukaryotic and eubacterial sources (52-57% similarity, 30-37% identity) and signature domains containing the Ser-Asp-His triad, which is characteristic of this family of proteases, were present. Northern blot experiments revealed the presence of an approx. 2.0-kb transcript in P. furiosus extracts, corresponding in length to that expected from mlr-2 expression. Initiation of transcription occurred 23 bp downstream from a putative BoxA promoter element.

Amino Acid Sequence↗
PubMed1995

DNA damage produced by exposure of supercoiled plasmid DNA to high- and low-LET ionizing radiation: effects of hydroxyl radical quenchers.

Production of DNA damage by exposure to ionizing radiation was measured in two in vitro systems. A supercoiled plasmid of 7.3 kbp was isolated and exposed in an aqueous environment to 60Co gamma rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (ssbs) and double-strand breaks (dsbs) were computed from the conversion of the supercoil to its relaxed and linear forms. The relative effectiveness (neutrons:gamma-rays) for destruction of genetic transforming activity of M13 viral DNA was 0.23, close to that for ssb production, in contrast with the situation for biological effects such as lethality, mutagenesis and cellular transformation measured in mammalian cells, where RBEs are > 1. The role of hydroxyl (OH) radicals in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both ssbs and dsbs. These findings are consistent with a model for ssb and dsb induction by high linear energy transfer radiation that involves OH radical medication.

Acetates↗
PubMed1995

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.

Amino Acid Sequence↗
PubMed1995

Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.

Archaea↗
PubMed1995

Rapid differentiation of hyperthermophilic Archaea by restriction mapping of the intergenic spacer regions of the ribosomal RNA operons.

We report a sensitive and convenient method for rapid differentiation of new isolates of hyperthermophilic Archaea. Polymerase chain reaction (PCR) was used to amplify the intergenic spacer regions of the ribosomal RNA operons of eight Archaea. Spacer regions from one Euryarcheote, Pyrococcus furiosus, and one Crenarcheote, Pyrodictium brockii, were sequenced completely. Restriction fragment length polymorphism (RFLP) analyses were performed on the spacer regions from eight hyperthermophilic Archaea, and the restriction patterns were used as fingerprints for six known strains and two isolates. The PCR-RFLP method used in this study allowed the differentiation of seven of the eight strains tested and could be generally applicable to all the Archaea.

Archaea↗
PubMed1994

Isolation of maltose-regulated genes from the hyperthermophilic archaeum, Pyrococcus furiosus, by subtractive hybridization.

The hyperthermophilic archaeum, Pyrococcus furiosus, utilizes maltose as a preferred carbon source for growth. 32P-labeled complementary DNA (cDNA) probes representing maltose-regulated genes were obtained by a subtractive hybridization procedure that minimized retrieval of ribosomal RNA (rRNA) sequences during screening. Genomic DNA clones were isolated by positive hybridization to these probes. Genes whose expression varied both in the level of transcription, relative to rRNA, as well as in the degree of regulation were obtained; the extent of regulation varied over a wide range, from as little as fivefold to as high as 50-100-fold. DNA sequence analysis of several of these regulated genes indicated that the subtraction library included gene products required for maltose utilization (e.g., pyruvate dikinase), as well as growth-rate-related genes such as those encoding ribosomal proteins and RNA polymerase subunits. Our approach is applicable to studying gene regulation in organisms that are not amenable to classical genetic techniques.

Archaea↗