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.     

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.     

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.     

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.     

Crystal structure of a novel zinc-binding ATP sulfurylase from Thermus thermophilus HB8

ATP sulfurylase (ATPS) is a ubiquitous enzyme that catalyzes the transfer of the adenylyl group from ATP to inorganic sulfate, producing adenosine 5'-phosphosulfate (APS) and pyrophosphate. The crystal structure of ATPS from Thermus thermophilus HB8 (TtATPS, 347 amino acid residues) in complex with APS was determined at 2.5 A resolution. TtATPS is composed of three domains [domain I (residues 1-134), domain II (residues 135-290), and domain III (residues 291-347)], like the Riftia pachyptila symbiont ATPS, but lacks a fourth domain present in ATPSs from the yeast Saccharomyces cerevisiae and from the fungus Penicillium chrysogenum. TtATPS forms a dimer in the crystal, and the manner of subunit association is different from that observed in dimeric R. pachyptila symbiont ATPS and in the hexameric S. cerevisiae and P. chrysogenum ATPSs. APS is located in the active site of TtATPS, which contains several motifs (QXRN, HXXH, and GRD) conserved in ATPSs. Unexpectedly, TtATPS binds one metal ion per subunit in domain III. XAFS measurement of the crystal and the Bijvoet difference Fourier map unambiguously characterized the metal ion as a zinc ion. The zinc ion is tetrahedrally coordinated by Cys294, Cys297, Cys306, and His310, and could not be removed from the protein by treatment with EDTA. The zinc ion binding site is far from the active site. Because all four residues coordinated to the zinc ion are conserved in the ATPSs from thermophilic bacteria such as Archaeoglobus fulgidus, Pyrococcus abyssi, and Sulfolobus solfataricus, zinc ion chelation may contribute to the thermal stability of these ATPSs.     

Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.     

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.     

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties.     

ATP-induced structural change of dephosphocoenzyme A kinase from Thermus thermophilus HB8

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.     

A hyperthermophilic laccase from Thermus thermophilus HB27

A copper-inducible laccase activity was detected in Thermus thermophilus HB27. The enzyme was partially purified and separated by SDS-PAGE. After staining, a gel slice containing a ~53-kDa protein was excised and treated with trypsin, and the in-gel digests were analyzed by mass spectrometry. By mass fingerprinting, the peptides were found to share identity with the TTC1370 protein of the thermophile, which was tentatively annotated as a laccase in the whole genome analysis, albeit experimental evidence was lacking. The assigned mass nearest to the N-terminal sequence was that from Gln²³ to Lys³¹. By signal peptide prediction, TTC1370 protein was assumed to be a secretory protein starting from Gln²³. The DNA encoding the mature protein was then cloned and expressed in Escherichia coli. The recombinant enzyme, expressed as an apoprotein, was dialyzed against copper-containing buffer to yield a holoprotein. The holoprotein was purified to homogeneity, which displayed a blue color typical of laccases and oxidized canonical laccase substrates such as guaiacol and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The enzyme was most notable for its striking thermophilicity; the optimal reaction temperature was ~92°C and the half-life of thermal inactivation at 80°C was >14 h, ranking it as the most thermophilic laccase reported thus far.     

Molecular multiplicity of nuclease TT1 from Thermus thermophilus HB8

Purified nuclease TT1 from Thermus thermophilus HB8 has multimolecular weight forms, each of which is composed of three different subunits, alpha (10.8 x 10(4)), beta (7.8 x 10(4)), and gamma (4.1 x 10(4)). The molecular weights of this enzyme were estimated by gel filtration, polyacrylamide gel electrophoresis and equilibrium sedimentation. It was found that most of the enzyme has a molecular weight of about 22 x 10(4) being a monomer having the subunit composition of alpha beta gamma. The remaining part of the enzyme has larger molecular weights and is considered to be size-isomers of alpha beta gamma. The alpha-helical content, 5.5--6.5%, and the beta-structure, about 28%, were estimated from the CD spectrum at 4 degrees C.     

Substrate specificity of nuclease TT1 from Thermus thermophilus HB8

The substrate and the action mechanism of a nuclease named nuclease TT1, from the culture broth of an extreme thermophile, Thermus thermophilus HB8, were investigated. The enzyme is nonspecific for the sugar moiety and cleaves both single- and double-stranded DNAs, rRNA, tRNA and oligonucleotides irrespective of chain length to produce 5'-mononucleotides exonucleolitically. The action mechanism is processive and the enzyme shows no porality of degradation. The minimal unit as a substrate is a 5'-dinucleotide. The rate of hydrolysis is independent of a terminal phosphate group. The substrate lacking a 5'-phosphoryl group is degraded to leave the 5'-terminus and the penultimate nucleotide (NpN) as a core. The substrate possessing a 3'-phosphoryl group is degraded to leave the mononucleoside 5',3'-diphosphates (pNp). However, NpN and pNp are gradually degraded by a large dose of the enzyme to produce a 5'-mononucleotide. The enzyme is free from nonspecific phosphatase and phosphodiesterase activities. Application of this enzyme to determine the sequence of oligonucleotides is shown.     

Crystal structures of possible lysine decarboxylases from Thermus thermophilus HB8

TT1887 and TT1465 from Thermus thermophilus HB8 are conserved hypothetical proteins, and are annotated as possible lysine decarboxylases in the Pfam database. Here we report the crystal structures of TT1887 and TT1465 at 1.8 A and 2.2 A resolutions, respectively, as determined by the multiwavelength anomalous dispersion (MAD) method. TT1887 is a homotetramer, while TT1465 is a homohexamer in the crystal and in solution. The structures of the TT1887 and TT1465 monomers contain single domains with the Rossmann fold, comprising six alpha helices and seven beta strands, and are quite similar to each other. The major structural differences exist in the N terminus of TT1465, where there are two additional alpha helices. A comparison of the structures revealed the elements that are responsible for the different oligomerization modes. The distributions of the electrostatic potential on the solvent-accessible surfaces suggested putative active sites.     

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.     

Cytochrome oxidase from an extreme thermophile. Thermus thermophilus HB8

The cytochrome oxidase (EC 1.9.3.1) of $\text{Thermus}\text{thermophilus}$ HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme $\text{a}$ and heme $\text{c}$ as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with $\text{T}\text{.}\text{thermophilus}$ cytochrome c-552. The oxidation of $\text{T}\text{.}\text{thermophilus}$ cytochrome $\text{c}$-555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome $\text{c}$-552. The enzyme was highly stable to heat.     

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.