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At least 91 records · Page 5Linked to original sources

PubMed2007

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring.

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection.

Archaeal Proteins↗

PubMed2006

Contribution of the C-terminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS-1.

Chaperonin is a double ring-shaped oligomeric protein complex, which captures a protein in the folding intermediate state and assists its folding in an ATP-dependent manner. The chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, is a group II chaperonin and is composed of two distinct subunits, alpha and beta. Although these subunits are highly homologous in sequence, the homo-oligomer of the beta-subunit is more thermostable than that of the alpha-subunit. To identify the region responsible for this difference in thermostability, we constructed domain-exchange mutants. The mutants containing the equatorial domain of the beta-subunit were more resistant to thermal dissociation than the mutants with that of the alpha-subunit. Thermostability of a beta-subunit mutant whose C-terminal 22 residues were replaced with those of the alpha-subunit decreased to the comparable level of that of the alpha-subunit homo-oligomer. These results indicate that the difference in thermostability between alpha- and beta-subunits mainly originates in the C-terminal residues in the equatorial domain, only where they exhibit substantial sequence difference.

Amino Acid Sequence↗

PubMed1999

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii.

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 Umg(-1). The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent Km values for ADP and fructose-6-phosphate were 0.6mM (apparent Vmax of 243Umg(-1)) and 1.47mM (apparent Vmax of 197Umg(-1)), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na+ and K+. The protein had a subunit size of 42.2kDa and an estimated native molecular weight of 66kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5-8.0, and the apparent Km values for fructose-1,6-bisphosphate and AMP were 0.56mM (apparent Vmax of 2.9Umg(-1) and 12.5mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases.

Adenosine Diphosphate↗

PubMed1999

The first description of an archaeal hemicellulase: the xylanase from Thermococcus zilligii strain AN1.

A xylanase has been found in the archaeon Thermococcus zilligii strain AN1 (DSM 2770), which grows optimally at 75 degrees C. The enzyme had a molecular mass of 95 kDa and a unique N-terminal sequence. It had activity against all five xylans tested and against xylose oligomers, but not against other carbohydrate polymers. The K(m) values found for xylans were typical of those found for bacterial xylanases. The pH optimum for activity was pH 6, and the enzyme half-life at 100 degrees C was 8 min. This is the first description of any archaeal hemicellulase.

Amino Acid Sequence↗

PubMed1999

A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure.

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85 degrees C (Topt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%-93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that Phabitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained.

Amino Acid Sequence↗

PubMed2000

Cloning, expression, and characterization of pyrrolidone carboxyl peptidase from the archaeon Thermococcus litoralis.

The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and characterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried out on enzyme substrate specificity and stability.

Amino Acid Sequence↗

PubMed2002

A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations.

A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli. The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85 degrees C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70 degrees C. Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity. The enzyme was partially inhibited by EDTA and p-hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity. Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids. preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine.

Amidohydrolases↗

PubMed1998

Lipids of Thermococcus hydrothermalis, an archaea isolated from a deep-sea hydrothermal vent.

The membrane lipids of a deep-sea hydrothermal vent archaea, Thermococcus hydrothermalis, were isolated, purified, and structurally characterized. On the basis of acid methanolysis and spectroscopic studies, the polar lipids, amounting to 4.5% (w/w) of the dry cells, comprised diphytanyl glycerol diethers and dibiphytanyldiglycerol tetraethers, in a 45:55 ratio. No cyclopentane ring was present in the tetraethers. From the neutral lipids, accounting for 0.4% (w/w) of the dry cells, besides low amounts of di- and tetraethers occurring in a free form, four acyclic tetraterpenoid hydrocarbons, di- and tri-unsaturated were identified. All were structurally related to lycopane. The presence of these hydrocarbons provides some evidence that lycopane, widely distributed in oceans, could be derived, at least partially, from the hydrocarbons synthesized by some thermophilic Archaea. Finally, analysis of the uninoculated culture medium indicates that fatty acid derivatives and some steroid and triterpenoid compounds identified in the lipidic extract of the archaea originated from the culture medium.

Archaea↗

PubMed2005

Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis.

Enzymatic and thermodynamic characteristics of type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (Tk-IDI) from Thermococcus kodakaraensis, which catalyzes the interconversion of IPP and DMAPP, were examined. FMN was tightly bound to Tk-IDI, and the enzyme required NADPH and Mg2+ for the isomerization in both directions. The melting temperature (Tm), the change of enthalpy (deltaH(m)), and the heat capacity change (deltaC(p)) of Tk-IDI were 88.0 degrees C, 444 kJ mol(-1), and 13.2 kJ mol(-1) K(-1), respectively, indicating that Tk-IDI is fairly thermostable. Kinetic parameters dramatically changed when the temperature crossed 80 degrees C even though its native overall structure was stably maintained up to 90 degrees C, suggesting that local conformational change would occur around 80 degrees C. This speculation was supported by the result of the circular dichroism analysis that showed the shift of the alpha-helical content occurred at 80 degrees C.

Amino Acid Sequence↗

PubMed2004

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans.

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.

Adenosine Triphosphate↗

PubMed2005

Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1.

The hydrogen (H2) production potential of the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 was evaluated at 85 degrees C. In batch cultivation using a complex medium supplemented with elemental sulfur (S0), evolution of H2S and CO2 was observed in the gas phase. When S0 was omitted and pyruvate or starch was added in the medium, the cells produced H2 at high levels instead of H2S. As the level of H2 appeared to correlate with the specific growth rate, analysis in continuous cultures was performed to develop a continuous H2 production system. In a steady-state condition at a dilution rate of 0.2 h-1, a continuous H2 production rate (per gram dry weight, gdw) of 24.9 and 14.0 mmol gdw-1 h-1 was observed in media supplemented with pyruvate and starch, respectively. In both cultivations, a high accumulation of acetate and alanine was found as metabolites. When the dilution rates were elevated in the medium with pyruvate, steady-state growth was observed up to 0.8 h-1, and a maximum H2 production rate of 59.6 mmol gdw-1 h-1 was obtained. Based on the experimental results along with data of the entire genome sequence, the metabolic pathway of the strain relating to starch and pyruvate degradation is discussed.

Amino Acids↗

PubMed2004

Crystal structures of the group II chaperonin from Thermococcus strain KS-1: steric hindrance by the substituted amino acid, and inter-subunit rearrangement between two crystal forms.

The crystal structures of the group II chaperonins consisting of the alpha subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native alpha8beta8-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL14-GroES7-(ADP)7 complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.

Adenosine Triphosphate↗

PubMedDate not supplied

Optimisation of growth conditions for continuous culture of the hyperthermophilic archaeon Thermococcus hydrothermalis and development of sulphur-free defined and minimal media.

The hyperthermophilic archaeon Thermococcus hydrothermalis was cultivated in continuous culture in a gas-lift bioreactor in the absence of elemental sulphur on both proteinaceous and maltose-containing media. Optimal conditions (pH, temperature and gas flow rate), determined on complex media that yielded maximal growth rate and maximal steady state cell density, were obtained at 80 degrees C, pH 6 and gas sparging at 0.2 v v(-1) min(-1). Higher steady state cell densities were obtained on a medium containing maltose and yeast extract. In order to design a defined and minimal media, the nutritional requirements of T. hydrothermalis were then investigated using continuous culture in the absence of elemental sulphur in the gas-lift bioreactor. First, the complex nutriments were replaced and a defined medium containing maltose, 19 amino acids and the two nitrogenous bases adenine and thymine, was determined. Secondly, selective feedings and withdrawal of amino acids showed requirements for 14 amino acids.

Adenine↗

PubMed2002

Tk-PTP, protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins.

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.

Amino Acid Sequence↗

PubMed2002

Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS-1 with reference to the ATP-dependent protein folding cycle.

The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.

Adenosine Diphosphate↗

PubMed2003

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis.

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.

Amino Acid Sequence↗

PubMed1999

High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS.

The Thermococcus litoralis 4-alpha-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coli resulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNA(AGA), tRNA(AGG) and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold. This new E. coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

Chaperonin 60↗

PubMed1999

Cloning of an alpha-glucosidase gene from Thermococcus hydrothermalis by functional complementation of a Saccharomyces cerevisiae mal11 mutant strain.

alpha-Glucosidase is found in methanogenic and thermophilic archaea and also in eukaryotes and bacteria. The gene encoding the enzyme was cloned from Thermococcus hydrothermalis by complementation of a Saccharomyces cerevisiae deficiency maltase mutant strain. The gDNA clone isolated encodes an open reading frame corresponding to a protein of 242 amino acids. The protein shows 42% identity to a Pyrococcus horikoshii unknown ORF but no similarities were obtained with polysaccharidase sequences.

Amino Acid Sequence↗