Thermoplasma Volcanium Research Articles

Oxyplasma meridianum gen. nov., sp. nov., an extremely acidophilic organotrophic member of the order Thermoplasmatales.

A mesophilic, hyperacidophilic archaeon, strain M1T, was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales. The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum, Thermoplasma volcanium and Thermogymnomonas acidicola, respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T=JCM 36570T) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales.

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 μM and 528 pmol/min/mg protein toward serotonin and 201 μM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice.

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 μM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.

Growth temperature and chromatinization in archaea

DNA in cells is associated with proteins that constrain its structure and affect DNA-templated processes including transcription and replication. HU and histones are the main constituents of chromatin in bacteria and eukaryotes, respectively, with few exceptions. Archaea, in contrast, have diverse repertoires of nucleoid-associated proteins (NAPs). To analyse the evolutionary and ecological drivers of this diversity, we combined a phylogenomic survey of known and predicted NAPs with quantitative proteomic data. We identify the Diaforarchaea as a hotbed of NAP gain and loss, and experimentally validate candidate NAPs in two members of this clade, Thermoplasma volcanium and Methanomassiliicoccus luminyensis. Proteomic analysis across a diverse sample of 19 archaea revealed that NAP investment varies from <0.03% to >5% of total protein. This variation is predicted by growth temperature. We propose that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix denaturation at higher temperatures, with implications for the origin of chromatin in both archaea and eukaryotes.

Structural studies of geranylgeranylglyceryl phosphate synthase, a prenyltransferase found in thermophilic Euryarchaeota.

Archaea are uniquely adapted to thrive in harsh environments, and one of these adaptations involves the archaeal membrane lipids, which are characterized by their isoprenoid alkyl chains connected via ether linkages to glycerol 1-phosphate. The membrane lipids of the thermophilic and acidophilic euryarchaeota Thermoplasma volcanium are exclusively glycerol dibiphytanyl glycerol tetraethers. The first committed step in the biosynthetic pathway of these archaeal lipids is the formation of the ether linkage between glycerol 1-phosphate and geranylgeranyl diphosphate, and is catalyzed by the enzyme geranylgeranylglyceryl phosphate synthase (GGGPS). The 1.72 Å resolution crystal structure of GGGPS from T. volcanium (TvGGGPS) in complex with glycerol and sulfate is reported here. The crystal structure reveals TvGGGPS to be a dimer, which is consistent with the absence of the aromatic anchor residue in helix α5a that is required for hexamerization in other GGGPS homologs; the hexameric quaternary structure in GGGPS is thought to provide thermostability. A phylogenetic analysis of the Euryarchaeota and a parallel ancestral state reconstruction investigated the relationship between optimal growth temperature and the ancestral sequences. The presence of an aromatic anchor residue is not explained by temperature as an ecological parameter. An examination of the active site of the TvGGGPS dimer revealed that it may be able to accommodate longer isoprenoid substrates, supporting an alternative pathway of isoprenoid membrane-lipid synthesis.

Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR–Cas Effector Complex

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR–Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1–Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA. Here we report the crystal structures of the Staphylococcus epidermidis Csm2 (SeCsm2) and Thermoplasma volcanium Csm3 (TvCsm3) at 2.4- and 2.7-Å resolutions, respectively. SeCsm2 adopts a monomeric globular fold by itself, in striking contrast to the previously reported Thermotoga maritima Csm2, which adopted an extended conformation and formed a dimeric structure. We propose that the globular monomeric form is the bona fide structure of Csm2. TvCsm3 forms a filamentous structure in the crystals. The molecular arrangement of TvCsm3 is similar to that of the stacked Cmr4 proteins in the Cmr complex, suggesting the functionally relevant architecture of the present Csm3 structure. We constructed model structures of the Csm complex, which revealed that Csm3 binds the crRNA and periodically deforms the crRNA-target duplex by a similar mechanism to that of Cmr4 in the Cmr complex. The model and mutational analysis suggest that the conserved lysine residue of Csm2 is important for target RNA binding, and Csm2 stabilizes the active structure of the Csm complex to facilitate the reaction.

Crystal Structure and Asymmetric Conformation of a K+ Channel RCK Domain

Regulator of conductance of K+ (RCK) domains govern K+ channel and transporter activity through binding of cytosolic nucleotides or ions, although the locations and ligand-selectivities of activation sites within these domains can vary. To gain further insight toward structures of activation sites and ligand-dependent conformational changes in RCK domains, we solved the crystal structure of the cytosolic RCK domain from a Ca2+-gated K+ channel cloned from Thermoplasma volcanium. Crystals were grown in the presence of Mg2+, an activator of the Thermoplasma K+ channel, and the structure was solved to 2.95 A resolution. The structure was found to contain two RCK domains per asymmetric unit, forming a homodimer. Interestingly, this homodimer was captured in an asymmetric state, and structural alignment of the chains with one another revealed a “hinge” that immediately followed the domain's N-terminal Rossmann fold region. This asymmetry suggested that the RCK dimer might either be in an intermediate, partially-liganded state, or (alternatively) that the asymmetric conformation represents the fully-liganded state. To determine the locations of divalent cation binding sites, the domain was co-crystallized with Ba2+. The Ba2+-bound structure yielded the same asymmetric conformation observed in the Mg2+ co-crystal, and the anomalous difference Fourier map revealed only one Ba2+ binding site, located at the RCK domain dimer interface and thus forming a metal bridge between adjacent subunits. Crystallization of the domain in a four-fold symmetrical “gating ring” form and small-angle X-ray scattering profiles obtained over a range of [Ca2+] are consistent with the idea that formation of the intersubunit metal bridge may drive a conformational change in the gating ring, which in turn may modulate channel gating.

Alba from Thermoplasma volcanium belongs to α-NAT's: An insight into the structural aspects of Tv Alba and its acetylation by Tv Ard1

The Alba superfamily proteins have been regarded as a conserved group of proteins in archaea and eukarya, which have shown to be important in nucleic acid binding, chromatic organization and gene regulation. These proteins often belong to the N-acetyltransferase (NAT) category (Nα-acetyltransferases or Nε-acetyltransferases) and undergo post-translational modifications. Here, we report the crystal structure of Alba from Thermoplasma volcanium (Tv Alba) at 2.4 Å resolution. The acetylation of Tv Alba was monitored and the N-terminal of Tv Alba has been shown to interact with acetyl coenzyme A (Ac-CoA). The chemical shift perturbation experiments of Tv Alba were performed in the presence of Ac-CoA and/or Tv Ard1, another T. volcanium protein that treats Tv Alba as a substrate. To examine the DNA binding capabilities of Tv Alba alone and in the presence of Ac-CoA and/or Tv Ard1, EMSA experiments were carried out. It is shown that although Tv Alba binds to Ac-CoA, the acetylation of Tv Alba is not related with its binding to dsDNA, and the involvement of the N-terminus in Ac-CoA binding demonstrates that Tv Alba belongs to the Nα-acetyltransferase family.

Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range.

Two glucoamylase-like genes, TVN1315 and Ta0286, from the archaea Thermoplasma volcanium and T. acidophilum, respectively, were expressed in Escherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6 barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display Km values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.

Specificity of Fur Binding to the Oxidative Stress Response Gene Promoter in the Facultative Anaerobic Archaeon &lt;i&gt;Thermoplasma volcanium&lt;/i&gt;

The genome of the facultative anaerobic thermoacidophilic archaeon Thermoplasma volcanium contains the open-reading frames (ORFs) tvsod and tvogg, which are predicted to encode a putative superoxide dismutase and an 8-oxoguanine DNA glycosylase, respectively. Tvsod is immediately upstream of tvogg, and these two ORFs are aligned in a head-to-tail manner in a single operon. A previous study showed that T. volcanium contains an ORF (TVN0292) encoding the ferric uptake regulator (Fur) and that the T. volcanium Fur protein (TvFur) binds to its own promoter in a metal-dependent manner in vitro. Here, we demonstrated that TvFur also binds to the tvsod-tvogg promoter and determined the TvFur-binding sequences in the tvsod-tvogg promoter by DNaseI footprinting analysis. These results suggest that Fur is required for resistance against reactive oxygen species in this facultative anaerobic archaeon.

Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium

A gene from the thermophilic archaeon Thermoplasma volcanium encoding an L-threonine dehydrogenase (L-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr(137) was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: L-ThrDH-NAD(+), L-ThrDH in complex with NAD(+) and pyruvate, Y137F mutant in complex with NAD(+) and L-threonine, and Y137F in complex with NAD(+) and L-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like L-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via direct proton transfer driven by Tyr(137). The factors contributing to the thermostability of T. volcanium L-ThrDH were analyzed by comparing its structure to that of F. frigidimaris L-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium L-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like L-ThrDH.

Transcriptional factor Fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.

Improvement of protein stability and enzyme recovery under stress conditions by using a small HSP (tpv-HSP 14.3) from Thermoplasma volcanium

In this study we cloned and expressed a small heat shock protein, tpv-HSP 14.3, from thermoacidophilic archaeon Thermoplasma volcanium. This novel recombinant small heat shock protein was purified to homogeneity and produced a protein band of 14.3kDa on SDS-polyacrylamide gel. Transmission electron microscopy images of the negatively stained tpv-HSP 14.3 samples showed spherical particles of 13nm diameter. E. coli cells over expressing tpv-HSP 14.3 endowed the cells with some degree of thermotolerance. After exposure to 52°C for 120min, survivability of the E. coli cells expressing tpv-HSP 14.3 was approximately 2.5-fold higher than the control cells. As a molecular chaperone tpv-HSP 14.3 enhanced the thermal stabilization of substrate proteins, pig heart citrate synthase and bovine l-glutamic dehdyrogenase, considerably. The highest protection effect of tpv-HSP 14.3 was observed at 47°C for pig heart citrate synthase; the remaining activity was 5-fold higher than that of the sample without tpv-HSP 14.3. The tpv-sHSP 14.3 prevented inactivation of bovine l-glutamic dehdyrogenase the most effectively at 53°C; the residual activity was approximately 2-fold higher than that of the sample heated without tpv-HSP 14.3. However, refolding activity of the tpv-HSP 14.3 was relatively weak for the chemically denatured substrate proteins.

The Structures of Thermoplasma volcanium Phosphoribosyl Pyrophosphate Synthetase Bound to Ribose-5-Phosphate and ATP Analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium ( Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2 1. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP–Mg 2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.

Phylogenetic Analysis of Methanogens in the Pig Feces

In order to assess methanogen diversity in feces of pigs, archaeal 16S rRNA gene clone libraries were constructed from feces of the pig. After the amplification by PCR using primers Met86F and Met1340R, equal quantities of PCR products from each of the five pigs were mixed together and used to construct the library. Sequence analysis showed that the 74 clones were divided into ten phylotypes as defined by RFLP analysis. Phylogenetic analysis showed that three phylotypes were most closely affiliated with the genus Methanobrevibacter (46% of clones). The library comprised 55.4% unidentified euryarchaeal clones. Three phylotypes (LMG4, LMG6, LMG8) were not closely related to any known Euryarchaeota sequences. The phylogenetic analysis indicated that the archaea found in the libraries were all clustered into the Euryarchaeota. The data from the phylogenetic tree showed that those sequences belonged to three monophyletic groups. Phylotypes LGM2 and LGM7 grouped within the genus Methanobrevibacter. Phylotypes LGM4, LGM6, LGM8 and LGM9 grouped within the genus Methanosphaera. Other phylotypes grouped together, and formed a distantly related sister group to Aciduliprofundum boonei and species of the Thermoplasmatales including Thermoplasma volcanium and Thermoplasm acidophilum. Our results showed that methanogens belonging to the genus Methanobrevibacter were predominant in pig feces, and that many unique unknown archaea sequences were also found in the library. Nevertheless, whether these unique sequences represent new taxonomic groups and their role in the pig gut need further investigation.

A reported archaeal mechanosensitive channel is a structural homolog of MarR-like transcriptional regulators

Several archaeal mechanosensitive (MS) channels have been reported, including one from Thermoplasma volcanium designated MscTV. Here, we report the crystal structure of MscTV at 1.6-A resolution. Unexpectedly, MscTV was found to be a water-soluble protein exhibiting a winged helix-turn-helix (wHTH) motif, which is the signature of the MarR (multiple antibiotic resistance regulator) family of transcriptional regulators. A cell-based osmotic downshock functional assay demonstrated that MscTV was unable to protect a knockout strain of Escherichia coli from hypoosmotic shock, further indicating that it does not function as a MS channel. We propose this protein be renamed MLPTv for MarR-like protein from T. volcanium.

Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli

In this study, a gene coding for thermophilic serine protease of the ClpP class from the thermoacidophilic archaeon Thermoplasma volcanium (Tpv) was cloned and expressed in Escherichia coli. The primary sequence and domain analysis of this enzyme showed similarities (50-60% similarity) to signal peptide peptidases (SppA) of bacteria and other archaea. An increase of about tenfold in the activity was achieved by overexpression of Tpv SppA in E. coli, as detected by enzyme assays conducted using Ala-Ala-Phe-pNa and N-Suc-Ala-Ala-Pro-Phe-pNA as substrates. The recombinant enzyme, purified using an anion exchange column chromatography, displayed an apparent molecular mass of 26 kDa on SDS-PAGE analysis. Purified Tpv SppA was active in a broad range of pH and temperature with maximal activity at 60 degrees C and between pH 7.5 and pH 8.0. The activity of the enzyme was strongly inhibited by inhibitors typical for serine proteases, i.e., chymostatin and PMSF. The activity of the Tpv SppA and the stability at high temperature were significantly enhanced in the presence of 5 mM Ca(2+) ions. Our multiple sequence alignment data revealed a conserved Ser/Lys catalytic dyad in Tpv SppA that comprised Ser76 (nucleophile) and Lys128 (general base) residues. A search for a transmembrane domain using automated programs did not predict any signal peptide associated with the Tpv SppA and, therefore, suggested a cytoplasmic location for this enzyme.

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases--a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archaeons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.

The stability of the archaeal HU histone-like DNA-binding protein from Thermoplasma volcanium

The complete genome analysis of the archaeon Thermoplasma volcanium has revealed a gene assigned to encode the histone-like DNA-binding protein HU. Thermoplasma volcanium is a moderate thermophile growing around 60 degrees C and it is adaptable to aerobic and anaerobic environment and therefore it is unique as a candidate for the origin of eukaryotic nuclei in the endosymbiosis hypothesis. The HU protein is the major component of the bacterial nuclei and therefore it is an important protein to be studied. The gene for HUTvo protein (huptvo) was cloned from the genomic DNA of T. volcanium and overexpressed in Escherichia coli. A fast and efficient purification scheme was established to produce an adequate amount of bioactive protein for biochemical and biophysical studies. Highly purified HUTvo was studied for its DNA-binding activity and thermostability. As studied by circular dichroism and high-precision differential scanning microcalorimetry, the thermal unfolding of HUTvo protein is reversible and can be well described by a two-state model with dissociation of the native dimeric state into denatured monomers. The G versus T profile for HUTvo compared to the hyperthermophilic marine eubacterial counterpart from Thermotoga maritima, HUTmar, clearly shows that the archaeal protein has adopted a less efficient molecular mechanism to cope with high temperature. The molecular basis of this phenomenon is discussed.