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PubMed2005

Determination of coenzyme A levels in Pyrococcus furiosus and other Archaea: implications for a general role for coenzyme A in thermophiles.

Physiologically significant levels of intracellular coenzyme A were identified in Pyrococcus furiosus, Thermococcus litoralis, and Sulfolobus solfataricus, suggesting a role for CoA as an important low molecular mass thiol in the thermophilic Archaea. In P. furiosus, cells grown in the presence of sulfur showed significantly higher levels of oxidized CoA compared with those grown in the absence of S(0). T. litoralis showed strikingly similar CoA levels, although with low disulfide levels in both the presence and absence of S(0). S. solfataricus showed similarly high levels of CoA thiol, with correspondingly low levels of the CoA disulfide. These results are consistent with the identification of a coenzyme A disulfide reductase (CoADR) in P. furiosus and horikoshii as well as the presence of CoADR homologues in the genomes of S. solfataricus and T. kodakaraensis.

Amino Acid Sequence↗

PubMed2006

Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon.

The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (delta3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc(Tk) gene into strain delta3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc(Tk) displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO2 fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc(Tk). These results demonstrated that the Type III Rbc(Tk) from a hyperthermophile could support CO2 fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity.

Cell Proliferation↗

PubMed2005

Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein l30e.

The origin of reduced heat capacity change of unfolding (DeltaC(p)) commonly observed in thermophilic proteins is controversial. The established theory that DeltaC(p) is correlated with change of solvent-accessible surface area cannot account for the large differences in DeltaC(p) observed for thermophilic and mesophilic homologous proteins, which are very similar in structures. We have determined the protein stability curves, which describe the temperature dependency of the free energy change of unfolding, for a thermophilic ribosomal protein L30e from Thermococcus celer, and its mesophilic homologue from yeast. Values of DeltaC(p), obtained by fitting the free energy change of unfolding to the Gibbs-Helmholtz equation, were 5.3 kJ mol(-1) K(-1) and 10.5 kJ mol(-1) K(-1) for T.celer and yeast L30e, respectively. We have created six charge-to-neutral mutants of T.celer L30e. Removal of charges at Glu6, Lys9, and Arg92 decreased the melting temperatures of T.celer L30e by approximately 3-9 degrees C, and the differences in melting temperatures were smaller with increasing concentration of salt. These results suggest that these mutations destabilize T.celer L30e by disrupting favorable electrostatic interactions. To determine whether electrostatic interactions contribute to the reduced DeltaC(p) of the thermophilic protein, we have determined DeltaC(p) for wild-type and mutant T.celer L30e by Gibbs-Helmholtz and by van't Hoff analyses. A concomitant increase in DeltaC(p) was observed for those charge-to-neutral mutants that destabilize T.celer L30e by removing favorable electrostatic interactions. The crystal structures of K9A, E90A, and R92A, were determined, and no structural change was observed. Taken together, our results support the conclusion that electrostatic interactions contribute to the reduced DeltaC(p) of T.celer L30e.

Archaeal Proteins↗

PubMed2005

Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases.

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.

Amino Acid Sequence↗

PubMed2002

Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII.

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.

Bacillus subtilis↗

PubMed2002

Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant.

We examined the molecular basis of ddNTP selectivity in archaeal family B DNA polymerases by randomly mutagenizing the gene encoding Thermococcus sp. JDF-3 DNA polymerase and screening mutant libraries for improved ddNTP incorporation. We identified two mutations, P410L and A485T, that improved ddNTP uptake, suggesting the contribution of P410 and A485 to ddNTP/dNTP selectivity in archaeal DNA polymerases. The importance of A485 was identified previously in mutagenesis studies employing Pfu (A486) and Vent (A488) DNA polymerases, while the contribution of P410 to ddNTP/dNTP selectivity has not been reported. We demonstrate that a combination of mutations (P410L/A485T) has an additive effect in improving ddNTP incorporation by a total of 250-fold. To assess the usefulness of the JDF-3 P410L/A485T in fluorescent-sequencing applications, we compared the archaeal mutant to Taq F667Y with respect to fidelity and kinetic parameters for DNA and dye-ddNTPs. Although the Taq F667Y and JDF-3 P410L/A485T mutants exhibit similar K(m) and V(max) values for dye-ddNTPs in single-base extension assays, the archaeal mutant exhibits higher fidelity due to a reduced tendency to form certain (ddG:dT, ddT:dC) mispairs. DNA polymerases exhibiting higher insertion fidelity are expected to provide greater accuracy in SNP frequency determinations by single-base extension and in multiplex minisequencing assays.

Base Sequence↗

PubMed2000

Identification of archaeal genes encoding a novel stationary phase-response protein.

A novel stationary phase-response protein has been identified in the acid-soluble protein extract of the thermophilic archaeon, Thermococcus zilligii. N-Terminal sequencing data were used to identify likely genes for homologues of the protein in the complete genome sequences of various archaeal species. The corresponding genes were identified and analysed. The genes encode a protein ranging from 83 to 92 amino acids in length, with a calculated pI ranging from 4.6 to 9.7. The amino acid sequences of the genes were highly conserved, even between members belonging to the different archaeal kingdoms. The computed secondary structure of the protein indicates it consists of four large helical regions separated by short coiled regions. We propose this protein as a candidate regulator of gene expression in stationary phase.

Amino Acid Sequence↗

PubMed2001

Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond.

From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.

Amino Acid Sequence↗

PubMed2003

Surface histidine residue of archaeal histone affects DNA compaction and thermostability.

Archaeal histone, which possesses only the core domain part of eukaryal histone, induced DNA compaction by binding to DNA. Based on structural modeling, tetramer formation by dimer-dimer interaction is considered to require two intermolecular ion pairs formed between histidine and aspartate. To examine the role of the ion pairs on DNA compaction, mutant histones were constructed and analyzed using HpkB from Thermococcus kodakaraensis KOD1 as a model protein. The mutant histones, HpkB-H50A, HpkB-H50V, and HpkB-H50G were constructed by replacing conserved surface His50 with Ala, Val, and Gly, respectively. Circular dichroism analysis indicated no significant difference between wild-type and mutants in their structures. Gel mobility shift assays showed that all mutants possessed DNA binding ability, like wild-type HpkB, however all mutants compacted DNA less efficiently than the wild-type. Moreover, all mutants could not maintain the nucleosome-like structure (compacted form of DNA) above 80 degrees C. These results suggest that surface ion pairs between His and Asp play an important role in maintenance of nucleosome structure and DNA stabilization at high temperature.

Amino Acid Substitution↗

PubMed2001

Structural basis for the ADP-specificity of a novel glucokinase from a hyperthermophilic archaeon.

BACKGROUND: ATP is the most common phosphoryl group donor for kinases. However, certain hyperthermophilic archaea such as Thermococcus litoralis and Pyrococcus furiosus utilize unusual ADP-dependent glucokinases and phosphofructokinases in their glycolytic pathways. These ADP-dependent kinases are homologous to each other but show no sequence similarity to any of the hitherto known ATP-dependent enzymes. RESULTS: We solved the crystal structure at 2.3 A resolution of an ADP-dependent glucokinase from T. litoralis (tlGK) complexed with ADP. The overall structure can be divided into large and small alpha/beta domains, and the ADP molecule is buried in a shallow pocket in the large domain. Unexpectedly, the structure was similar to those of two ATP-dependent kinases, ribokinase and adenosine kinase. Comparison based on three-dimensional structure revealed that several motifs important both in structure and function are conserved, and the recognition of the alpha- and beta-phosphate of the ADP in the tlGK was almost identical with the recognition of the beta- and gamma-phosphate of ATP in these ATP-dependent kinases. CONCLUSIONS: Noticeable points of our study are the first structure of ADP-dependent kinase, the structural similarity to members of the ATP-dependent ribokinase family, its rare nucleotide specificity caused by a shift in nucleotide binding position by one phosphate unit, and identification of the residues that discriminate ADP- and ATP-dependence. The strict conservation of the binding site for the terminal and adjacent phosphate moieties suggests a common ancestral origin of both the ATP- and ADP-dependent kinases.

Adenosine Diphosphate↗

PubMed2001

Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry.

BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO2 fixation in plants, algae, and bacteria. Rubiscos have been so far classified into two types. Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L8S8), but type II is usually composed only of two L subunits (L2). Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea. RESULTS: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Rubisco) was determined at 2.8 A resolution. The enzyme is composed only of L subunits and showed a novel (L2)5 decameric structure. Compared to previously known type I enzymes, each L2 dimer is inclined approximately 16 degrees to form a toroid-shaped decamer with its unique L2-L2 interfaces. Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures. CONCLUSIONS: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L2)5 decamer. Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures. The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.

Amino Acid Sequence↗

PubMed1998

High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.

BACKGROUND: Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS: The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS: The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

Amino Acid Sequence↗

PubMed2004

Kinetically robust monomeric protein from a hyperthermophile.

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.

Archaeal Proteins↗

PubMed2006

A hyperthermophilic protein acquires function at the cost of stability.

Active-site residues are not often optimized for conformational stability (activity-stability trade-offs) in proteins from organisms that grow at moderate temperature. It is unknown if the activity-stability trade-offs can be applied to proteins from hyperthermophiles. Because enzymatic activity usually increases at higher temperature and hyperthermophilic proteins need high conformational stability, they might not sacrifice the stability for their activity. This study attempts to clarify the contribution of active-site residues to the conformational stability of a hyperthermophilic protein. We therefore examined the thermodynamic stability and enzymatic activity of wild-type and active-site mutant proteins (D7N, E8A, E8Q, D105A, and D135A) of ribonuclease HII from Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced denaturation was measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation were highly reversible in these proteins. All the mutations of these active-site residues, except that of Glu8 to Gln, reduced the enzymatic activity dramatically but increased the protein stability by 7.0 to 11.1 kJ mol(-1) at 50 degrees C. The mutation of Glu8 to Gln did not seriously affect the enzymatic activity and increased the stability only by 2.5 kJ mol(-1) at 50 degrees C. These results indicate that hyperthermophilic proteins also exhibit the activity-stability trade-offs. Therefore, the architectural mechanism for hyperthermophilic proteins is equivalent to that for proteins at normal temperature.

Amino Acid Substitution↗

PubMed2004

Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies.

Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.

Amyloid↗

PubMed2002

The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii.

The crystal structure of pyrrolidone-carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus furiosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefaciens. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors.

Amino Acid Sequence↗

PubMed1987

A possible biochemical missing link among archaebacteria.

Until recently all archaebacteria isolated conformed to one of three basic phenotypes: they were either methanogens, extreme halophiles, or ('sulphur-dependent') extreme thermophiles. However, a novel phenotype, that fits none of these categories, has recently been described. The organism, strain VC-16 (tentatively called "Archaeoglobus fulgidus") reduces sulphate--the only archaebacterium so far known to do so--and makes very small quantities of methane, although it lacks some of the cofactors normally associated with methanogenesis. These characteristics suggest that strain VC-16 might represent a transition form between an anaerobic thermophilic sulfur-based type of metabolism (which seems to be the ancestral metabolism for archaebacteria and methanogenesis (which somehow then derives from it). We here show that the lineage represented by strain VC-16 arises from the archaebacterial tree precisely where such an interpretation would predict that it would, between the Methanococcus lineage (which is the deepest of the methanogen branchings) and that of Thermococcus (the deepest of all branchings on the methanogen side of the tree).

Archaea↗

PubMed2004

Genetic elements of Thermococcales.

This minireview summarizes our current knowledge about archaeal genetic elements in the hyperthermophilic order Thermococcales in the phylum Euryarchaeota. This includes recent work on the first virus of Pyrococcus, PAV1, the discovery of novel unique virus morphotypes in hot deep-sea environments, and preliminary observations on novel cryptic plasmids. We also review the work accomplished over the last 5 years in the development of genetic tools for members of the Pyrococcus and Thermococcus genera, mainly in our laboratories.

Antigens, Archaeal↗