Expression,purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

The low-molecular-weight protein tyrosine phospha- tases （PTPase） exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phosphate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn^2＋, Mg^2＋, Ca^2＋, Ba^2＋, and Ni^2＋, but inhibited by Zn^2＋, Cu^2＋, Cl^-, and SO^2-. These results suggest that this heat-resistant PTPase may play important roles in vivo in the adaptation of the microorganism to extreme temperatures and specific nutritional conditions.     

Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8.

Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.     

Structural characterization of neutral and acidic glycolipids from Thermus thermophilus HB8

The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro, and the other, NGL-C, was Galf(β1-2)Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl(2)Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.     

S-layer protein from Thermus thermophilus HB8 assembles into porin-like structures

The cells of the extreme thermophile Thermus thermophilus are surounded by a regular layer (S-layer) built up by a protein with an apparent molecular mass of 100 kDa (P100). From purified membrane fractions, three different class of two-dimensional crystals can be obtained by following alternative extractive procedures. One of these crystals, with p6 symmetry, clearly represents the native S-layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins. We demonstrate here by limited protreolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein. Our biochemical data also show how this protein forms Ca²⁺-stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.     

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8.

Cell-free extracts of Thermus thermophilus HB8 catalyze the in vitro, NADH-dependent reduction of potassium tellurite (K2TeO3). Three different protein fractions with tellurite-reducing activities were identified. Two exhibited high molecular weight and were composed of at least two different polypeptides. The protein in the third fraction was purified to homogeneity and had a single polypeptide chain of 53 to 54 kilodaltons, with an isoelectric point of 8.1. Each enzyme was thermostable, the temperature optimum was 75 degrees C, and 30 mM NaCl, 1.5 M urea, or 0.004% sodium dodecyl sulfate caused 50% inhibition of the enzymes. However, 2% Triton X-100 did not have an inhibitory effect. The enzymes were also able to catalyze the reduction of sodium selenite and sodium sulfite in vitro. NADH was replaceable by NADPH. Divalent cations, such as Ca2+ and Ba2+, had no effect on the activity, while similar concentrations of Zn2+, Ni2+, and Cu2+ abolished the activity. This reductase activity could enable these bacteria both to reduce K2TeO3 and to increase their tolerance toward this salt.     

Crystal structure of ribosomal protein L27 from Thermus thermophilus HB8

Ribosomal protein L27 is located near the peptidyltransferase center at the interface of ribosomal subunits, and is important for ribosomal assembly and function. We report the crystal structure of ribosomal protein L27 from Thermus thermophilus HB8, which was determined by the multiwavelength anomalous dispersion method and refined to an R-factor of 19.7% (R(free) = 23.6%) at 2.8 A resolution. The overall fold is an all beta-sheet hybrid. It consists of two sets of four-stranded beta-sheets formed around a well-defined hydrophobic core, with a highly positive charge on the protein surface. The structure of ribosomal protein L27 from T. thermophilus HB8 in the RNA-free form is investigated, and its functional roles in the ribosomal subunit are discussed.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Crystal structure of the GTP-binding protein Obg from Thermus thermophilus HB8

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T.thermophilus and B.subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180 degrees, around the G domain axis. In the T.thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state.     

Solution structure of ribosomal protein L16 from Thermus thermophilus HB8

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an alpha+beta sandwich structure combined with two additional beta sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the C(alpha) model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics.     

Structure of peptidoglycan from Thermus thermophilus HB8.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.     

Phosphoenolpyruvate-insensitive phosphofructokinase isozyme from Thermus thermophilus HB8.

In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.     

Isolation and crystallization of phenylalanyl-tRNA-synthetase from Thermus thermophilus HB8

Method of isolation of phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 is described, including chromatography on DEAE-sepharose, ammonium sulfate fractionation, hydrofobic chromatography on Toyopearl, gel filtration on ultrogel AcA-34, chromatography on phenylalanylaminohexyl-sepharose and heparine-sepharose. Yield of the purified enzyme was 10 mg from 1 kg of T. thermophilus cells. The enzyme is found to consist of two types of subunits with molecular masses 92 and 36 kDa and is likely to be a tetramer protein with molecular mass 250 kDa. Crystals of phenylalanyl-tRNA synthetase suitable for X-ray structural studies have been obtained.     

Cloning,expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8

Abstract

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg²⁺. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90?°C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.     

Plasmid-associated aggregation in Thermus thermophilus HB8

Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.     

Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8

Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8. As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E. coli and B. stearothermophilus. Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T. thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not. The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another. The conformational transition involving the anticodon of E. coli tRNAGlu as complexed with Glu-tRNA synthetase from T. thermophilus is necessary for the aminoacylation activity.